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Full text of "Review of intramural research 1959"

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Review of 
INTRAMURAL RESEARCH 



NATIONAL INSTITUTES OF HEALTH 



1959 



U.S. DEPARTMENT OF 



HEALTH, EDUCATION, AND WELFARE 



Public Health Service 




Library 

National Institutes of H«W 

Bethesda, 14, Marylad 



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ISSUED IN A LIMITED EDITION FOR ADMINISTRATIVE USE 



Review of 



INTRAMURAL RESEARCH 



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NATIONAL INSTITUTES OF HEALTH 



1959 



U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 



PUBLIC HEALTH SERVICE 

NATIONAL INSTITUTES OF HEALTH 
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INSTITUTE RESEARCH DIRECTORS 



National Cancer Institute G. Burroughs Mider, M.D. 

Scientific Director 

Charles G. Zubrod, M.D. 
Clinical Director 

National Heart Institute Robert W. Berliner, M.D. 

Scientific Director 
National Institute op Arthritis 

and Metabolic Diseases DeWitt Stetten, M.D. 

Scientific Director 

Joseph J. Bunim, M.D. 
Clinical Director 
National Institute of Allergy 

and Infectious Diseases Dorland J. Davis, M.D. 

Scientific Director 

Vernon Knight, M.D. 
Clinical Director 

National Institute of Mental Health . . . Robert B. Livingston, M.D. 

Scientific Director 

Robert A. Cohen, M.D 
Clinical Director 
National Institute of Neurological 

Diseases and Blindness Robert B. Livingston, M.D 

Scientific Director 

G. Milton Shy, M.D 
Clinical Director 
National Institute of Dental 

Research Seymour Kreshover, M.D., D.D.S. 

Scientific Director 

Division of Biologics Standards Roderick Murray, M.D. 

Scientific Director 

Joseph E. Smadel, M.D., 
Associate Director for Intramural Research. 

Eli M. Nadel, M.D., 
Executive Secretary, Scientific and Clinical Directors Group. 



m 



FOREWORD 



The National Institutes of Health constitutes the principal research arm of 
the Public Health Service and is charged with the conduct and support of 
research, research training, and related activities. The research conducted by 
the National Institutes of Health is the subject of this publication. It is not, 
therefore, a comprehensive presentation of the program of the Public Health 
Service or of the National Institutes of Health. Bather, it is a series of reviews 
designed to provide acquaintance with what is familiarly known as NIH in- 
tramural research. 

Glimpses of the extent to which the intramural research effort has con- 
tributed to the advance of medical research are provided in the eight Annual 
Reports comprising this compendium. These reports, which received only 
minor editing, provide the reader with the flavor and philosophy of the sepa- 
rate Institute research programs as presented by their scientific leaders. Yet, 
in such a presentation, one may miss the thread of mission that ties each of the 
operating research organizations into the program of the National Institutes 
of Health. It is appropriate, therefore, to pursue this aspect briefly here. 

The mission of the National Institutes of Health as a whole and of its 
components is to develop the facilities, resources, and attitudes which would be 
most effective in acquiring new knowledge concerning disease processes, to 
the end of relieving suffering, bringing about cure and rehabilitation, and as- 
suring the prevention, whenever possible, of disease. Broadly considered, this 
mission involves providing the wherewithal and cultivating suitable soil for a 
systematic study of man and his milieu with the ultimate objective of contribut- 
ing to improved health. From this overall point of view, the NIH does not 
differentiate between what is done intramurally and extramurally. However, 
within these conceptions are contained more specific objectives only some of 
which can be sought for within an intramural research program, while the 
others may be searched out most expeditiously by support of work in other 
institutions through the extramural program. 

This first Annual Review of NIH Intramural Research provides evidence 
of the magnitude of the effort — both in breadth and depth — and of the type 
of achievements that have placed this installation in the forefront of research 
in the medical sciences. 



N^e.VT\24 Q.^^a 



VYl^. 



James A. Shannon, 

Director. 



PREFACE 



It has been customary in recent years for the intra- 
mural research activities of the National Institutes 
of Health to be summarized annually for the pur- 
pose of administrative review. The reports origi- 
nated with senior investigators, were consolidated 
and commented on by section chiefs and labora- 
tory chiefs, and assessed by the Clinical Directors 
and Scientific Directors before transmission to the 
Office of the Director, NIH. Since these annual 
program reviews were designed essentially to meet 
administrative needs, they were not reproduced in 
quantity and were distributed only among those 
scientists and scientist-administrators directly 
concerned. 

The present volume represents an elaboration 
of the former practice in one respect. Although 
all the preliminary steps remain the same, and 
although the annual program reviews continue to 
serve their established purposes, it is now pro- 
posed to make available on a limited basis that ma- 
terial which represents the scientific contributions 
of the intramural staff of each Institute. The 
present undertaking stems from the consensus 
that a number of scientific and institutional needs 
would be met if there were an annual publication 
summarizing the work that has been done within 
NIH's own laboratory and clinical facilities. 

Nineteen fifty-nine is the first experimental year 
for such a publication. Between these covers 
there have been brought together the 1959 annual 
reports of intramural research of the seven Insti- 
tutes and the Division of Biologies Standards: 
Four sections summarize the total intramural pro- 
grams of individual Institutes, one presents the 
combined basic laboratory research activity of the 
Mental Health and the Neurology Institutes, three 
separate reports describe the clinical work in pro- 
grams of the Mental Health, Neurology, and Ar- 
thritis Institutes, and one represents the research 
activities that complement the regulatory activi- 
ties of the Division of Biologies Standards. 

It should be pointed out, without apology, that 
the volume is a compendium of individual reports 
that have been assembled and included without 



appreciable editorial change. Just as the admin- 
istrative organizations of the Institutes vary, so 
these reports range from the simple summation of 
a group of intramural research projects to a broad 
philosophical treatise on the role of science in re- 
lation to the society it serves. The reports also 
vary in length and in the degree to which the 
Scientific and Clinical Directors wrote their own 
summations and interpretations, as contrasted 
with their use of materials submitted to them by 
the Laboratory and Branch Chiefs. It is antici- 
pated that these annual program reviews in fu- 
ture years will be more uniform both in length and 
in general approach. 

In their present form, however, the pertinent 
portions of the 1959 program reviews are deemed 
suitable for limited internal use among the senior 
administrative and scientific staff members of the 
Institutes and their Boards of Scientific Coun- 
selors, and our scientific colleagues in close asso- 
ciation with NIH intramural research. 

The reports focus attention on intramural re- 
search bearing directly on the missions of the sev- 
eral Institutes. Thus, they do not and cannot 
take into account the high quality and wide range 
of services that make it possible for the intramural 
research programs to be successfully carried for- 
ward. 

One such group of services, for example, is 
found in the Division of Research Services. In 
addition to its housekeeping functions related to 
the maintenance of 40 buildings on a 300-acre in- 
stallation where almost 8,000 persons devote their 
energy in one way or another to medical research, 
the Division provided innumerable supporting 
services to the thousand intramural scientists. 
Typical of these services are the provision of 
nearly a million small animals annually for use 
by the investigators at Bethesda; the design and 
construction of new laboratory equipment not 
available from commercial sources; the provision 
of computation and data processing equipment and 
of photographic and art services necessary for the 
documentation of experimental results and the 



VII 



VIII 



PREFACE 



publication of scientific data ; and the provision of 
a trained staff to assist the scientific community in 
analyzing its problems. 

Similarly, the Office of Administrative Manage- 
ment provides a broad spectrum of central services 
related to the business operations of a large re- 
search establishment — personnel, budget and fi- 
nance, purchase and supply, and so on. 

The Clinical Center, too, is a central service of 
a highly specialized nature. It provides and 
maintains a 500-bed hospital environment, with 
the staff and equipment and the organization to 
permit the clinical investigators of the various In- 
stitutes to undertake their studies on selected 
patients. 

While this document is devoted to the scientific 
product of the NIH laboratories and clinics, it 
must be remembered that the scientist not only 
draws upon but is in fact many times dependent 
upon others for the optimum conduct of his stud- 
ies. In most instances the researcher may have 
under his immediate supervision only two or three 
technicians and other supporting personnel. On 
the average, however, if he is a laboratory scientist, 
he has four persons serving him in supporting 
capacities elsewhere on the grounds; and if he is 
a clinical investigator, the number is ten. 

This preface to the 1959 annual program reviews 
of the Scientific Directors is an inappropriate 
place to attempt to describe the many cohesive 
factors of the intramural research environment 
which underlie the separate presentations. One 
thinks of the part played by NIH and its scientists 
in fostering national and international scientific 
meetings, of the contributions of NIH scientists 
to the scientific literature, of the role of NIH in 
fostering collaborative research — the number of 
items is legion. 

The primary mission of the intramural pro- 
gram at NIH is to obtain new information of im- 
portance to biology and medicine. Because re- 
search and teaching go hand in glove, however, an 
appreciable portion of the effort of the NIH staff 
is devoted to the teaching and training of young 
men and women in the methods and philosophy 
of scientific research. Most of this educational 
activity is centered around the time-honored 
preceptor-student relationship, in the laboratory 
and on the ward. More formal instruction is 
given, however, to the Clinical Associates and the 



Research Associates: the former are assisted in 
qualifying for their Specialty Boards, and the 
latter are assisted in acquiring detailed knowledge 
in disciplines which are inadequately emphasized 
in the usual medical curriculum for those who 
would subsequently pursue a career in research. 
The course work, for example, includes 145 hours 
in organic chemistry, physical chemistry (includ- 
ing quantum mechanics, mathematics, statistics, 
tracer methods, and instrumentation), and semi- 
nars in psychology, biochemistry, physiology, 
microbiology, and genetics. In addition, a num- 
ber of the NIH staff voluntarily assume some 
teaching responsibility in one or another of the 
neighboring universities, and many give individu- 
al lectures at academic institutions throughout the 
country. 

It should be noted that during the year covered 
by these reports of the Scientific Directors, a 
great deal of thought was devoted to plans for the 
future intramural research program. In part this 
was a necessary concomitant of the construction of 
new buildings on the Bethesda campus and the 
receipt of authority to acquire a farm in nearby 
Maryland. Of a more elective nature was the 
preparation by the Scientific Directors of a careful 
and comprehensive projection of how and where 
NIH intramural research should develop over the 
next decade; this projection was submitted as a 
report to the Director, NIH, in May 1959. In 
brief, the availability during calendar years 1960 
and 1961 of new buildings to house the Division of 
Biologies Standards, the National Institute of 
Dental Research, and a large portion of the ex- 
tramural operations of the NIH will permit a 
moderate expansion of the intramural program. 
This is the first time such a possibility has ap- 
proached actuality since the opening of the Clin- 
ical Center in 195/£ *?The space to be acquired will 
be used to relieve the grave congestion of certain 
research groups, and to expand and to initiate 
research in selected fields not now adequately cul- 
tivated. One of the guiding principles in the 
allocation and utilization of the new space will be 
to move those nonclinical research groups who 
now occupy space in the Clinical Center to one of 
the laboratory buildings. This will allow an in- 
creasing emphasis to be given to clinical investiga- 
tions at the NIH, which now provides an almost 
unique opportunity in the medical world for the 



PREFACE 



IX 



study of disease in man in an environment abun- 
dantly supplied with outstanding scientists in 
many basic disciplines. 

The combination of facilities, equipment, staff, 
and stable operating funds for NIH's intramural 
research programs represents both a challenging 
opportunity in scientific terms and a major re- 
sponsibility in social terms. During years of rapid 
growth and change in both the substance and the 
dimension of the sciences related to health, it has 
been a matter of pride that NIH has maintained 
the excellence, in terms of stature and produc- 



tivity, that characterized those scientists and lab- 
oratories of which it is the lineal descendant. And 
it is a matter of conviction that the tradition of 
excellence shall be maintained. 



ifoyk 




hd— 



Joseph E. Smadel, M.D. 

Associate Director. 



556044—60- 



CONTENTS 



Page 

Foreword vi 

Preface vn 

National Cancer Institute 1 

Introduction 1 

Causes of cancer 3 

Chemicals 3 

Mechanisms of carcinogenesis 5 

Viruses 6 

Other carcinogens 10 

Host factors in carcinogenesis 11 

Cancers and their properties 11 

New tumors 12 

Plasma cells and plasmacytomas 12 

Dissemination of cancer 14 

Polysaccharide research 16 

Protein and amino acid chemistry 18 

Nucleic acid research 20 

Endocrinology. 22 

Radiation biology 24 

Natural history of leukemias 26 

Anemia and related disorders 27 

Weight loss 28 

Tissue culture 28 

Cytochemistry and histochemistry 29 

Dermatology 30 

Surgical and virus treatment of cancers 31 

Virus treatment 32 

Pyridoxine deficiency 33 

Radiological treatment of cancers 33 

Chemotherapy of cancers 34 

Choriocarcinoma 35 

Endocrine therapy 36 

o, p'-DDD 36 

Screening activities 36 

Antileukemic compounds 38 

Alkylating agents 40 

Tetracyclines . 41 

Miscellaneous studies 41 

Service functions 42 

National Heart Institute 43 

Laboratory of cellular physiology and metabolism .... 43 

Cellular physiology 43 

Metabolism 46 

Basic physiology of fat absorption and fat 

transport 46 

Dietary and hormonal factors determining 

serum lipoprotein levels 47 

Metabolism of cholesterol and therapeutic agents 

useful in lowering serum cholesterol levels .... 48 



Page 
Hypoalbuminemia and responsible mechanisms. 48 
Mechanisms of protein synthesis and degrada- 
tion 49 

Structure of proteins and the nature of the 

clotting process 49 

Disturbed metabolism of lipids in nephrosis and 

the immunochemical mechanisms involved. ... 49 
Development of techniques for radioassay in the 

liquid scintillation spectrometer 50 

Enzymes 50 

The metabolism of heterocyclic compounds 50 

The metabolism of three-carbon compounds. ... 51 
The biochemistry of cellular differentiation and 

protein synthesis 52 

Anaerobic oxidative phosphorylation and elec- 
tron transport 53 

Nucleotide decomposition 53 

The metabolism of onium compounds 53 

The metabolism of isoprene derivatives 54 

The metabolism of amino acids 54 

Laboratory of chemistry of natural products 56 

Laboratory of chemical pharmacology 58 

Development of new drugs 58 

Biogenic amines 58 

Passage of drugs across membranes 60 

Drug metabolism 61 

Studies in biochemical behavior 61 

Studies with ascorbic acid 63 

Laboratory of technical development 63 

Gas chromatography 63 

Microanalysis by excitation in low-pressure helium 

discharge 64 

Errors in catheter-recording systems 64 

Computer for analysis of overlapping distributions . . 64 

Valvular regurgitation 64 

Flow-meter development 65 

Theoretical analysis of transport 65 

Phosphorescence analysis 65 

Ultrasonics 66 

Laboratory of cardiovascular physiology 66 

Studies on the heart 66 

Studies of circulatory regulation — exercise 69 

Abdominal pressoreceptors 69 

Studies of the callicrein system 69 

Reflexly induced changes in renal function 70 

Laboratory of kidney and electrolyte metabolism. ... 70 

Laboratory of clinical biochemistry 73 

Experimental Therapeutics 75 

Vasoactive substances 75 

Metabolism of amino acids in man 76 

Action and metabolism of drugs 76 

Cardiodynamics 77 

XI 



XII 



CONTENTS 



Page 
Laboratory of clinical biochemistry — Continued 

Clinical Endocrinology 79 

Steroid metabolism 79 

Studies of disturbed water metabolism 81 

Studies of calcium metabolism 81 

Studies of aortic permeability to large molecules. 82 

Surgery branch 82 

Gerontology branch 86 

Human physiology 86 

Psychological studies 87 

Basic biology 87 

National Institute of Abtheitis and Metabolic 

Diseases 91 

Basic research 91 

Introduction 91 

Laboratory of nutrition and endocrinology 92 

Nutrition 93 

Factor 3 93 

Vitamin E 93 

Vitamin B12 and folic acid 94 

Germ-free animals 95 

Protein 95 

Obesity 95 

Guinea pigs 96 

Rabbits 96 

Endocrinology 96 

Glucose tolerance factor 96 

Experimental diabetes 96 

Pituitary hormones 97 

Steroids 97 

Midbrain lesions and endocrine activity 97 

Laboratory of Pharmacology and Toxicology 97 

Shock and infection 97 

Calcium in nerve and muscle function 97 

Amine metabolism 98 

Acetylation of polyamines 98 

Metabolism of histidine and related compounds. 98 

Cholesterol synthesis 99 

Gramicidin J 99 

Enzyme activity and molecular structure 99 

Sulfur amino acids 99 

Antibody localization 99 

Laboratory of biochemistry and metabolism 99 

Carbohydrate metabolism 99 

Nucleic acids: structure and metabolism 100 

Steroid metabolism 101 

Gene-enzyme relationships in histidine biosyn- 
thesis 101 

Role of polyamines in neutralization of bacteri- 
ophage DNA 102 

Enzyme induction 102 

Enzymatic utilization of model compounds 102 

Coenzyme studies 102 

Laboratory of pathology and histochemistry 102 

Hematologic and genetic studies 102 

Experimental arthritis 103 

Infectious processes 103 



Page 

Stress and endocrine effects 104 

Histochemical studies 104 

Renal architecture 105 

Germ-free animals 105 

Human pathology 105 

Laboratory of physical biology 105 

Laboratory of chemistry 109 

Analgesics 109 

Carbohydrates HO 

Metabolites HO 

Steroids 112 

Office of mathematical research 113 

Concluding comments 114 

Clinical investigations 115 

Arthritis and rheumatism branch 116 

Studies of Sjogren's syndrome 117 

Epidemiology of rheumatoid- and osteo-arthritis . 117 
Mechanism of action of steriods at molecular 

level (in vitro) 117 

Effect of certain compounds on uric acid syn- 
thesis in man H8 

New uricosuric agents in gout 118 

Enzymic and clinical studies in inborn errors of 

metabolism other than gout 119 

Genetic polymorphisms in man and other ani- 
mals 120 

Biochemical studies in diseases of the gastro- 
intestinal tract 121 

Pediatric metabolism branch 122 

Proposed investigations in cystic fibrosis of the 

pancreas 122 

Proposed studies in intestinal malabsorption in 

children 123 

Proposed investigations in glycogen-storage 

diseases 123 

Clinical endocrinology branch 123 

Carbohydrate metabolism 123 

The thyroid 124 

Metabolic diseases branch 126 

Clinical hematological research (studies in blood 

diseases) 126 

Studies of human total energy metabolism 129 

Mineral metabolism studies 129 

National Institute of Mental Health 

and 
National Institute of Neueological Diseases 

and Blindness 131 

Introduction • 131 

Basic research (NIMH-NINDB) 150 

Laboratory of biophysics 150 

Laboratory of neuroanatomical sciences 151 

Section on development and regeneration 151 

Section on neurocytologj 153 

Section on experimental neuropathology 154 

Section on functional neuroanatomy 155 

Section on perinatal physiology 156 



CONTENTS 



XIII 



Page 
Basic research — (NIMH-NINDB) — Continued 

Laboratory of neurochemistry 159 

Section on physical chemistry. 160 

Section on lipid chemistry 161 

Laboratory of neurophysiology 162 

Laboratory of cellular pharmacology 164 

Technical development 166 

Clinical investigations — NIMH 168 

Introduction 168 

Clinical care 172 

Adult psychiatry branch 173 

Psychosomatic medicine 173 

Personality development 175 

Family studies 177 

Clinical neuropharmacology research center 179 

Psychiatry 180 

Chemical pharmacology 182 

Behavioral sciences 183 

Child research branch 185 

Survey of developments 185 

Research developments — tentative findings 186 

Individual therapy and psychopathology 186 

Problems of technique 187 

Problems in ego structure 187 

Milieu therapy and life-space interview 188 

Learning disturbances 189 

Behavioral measurement 190 

Biographical studies 193 

Laboratory of clinical science 194 

Schizophrenia 194 

Aging 196 

Experimental allergic encephalomyelitis 196 

Sleep 197 

Metabolism 197 

Enzymatic activities in blood 199 

Laboratory of psychology 199 

Aging 200 

Animal behavior 203 

Perception and learning 205 

Child development 206 

Personality 209 

Office of the chief 210 

Laboratory of socio-environmental studies 213 

Office of the chief 214 

Community and population studies 215 

Social development and family studies 215 

Social studies in therapeutic settings 217 

Addiction Research Center — NIMH 218 

Addictive properties of new analgesics 219 

Chronic and acute intoxication 221 

Biochemistry of addiction 222 

Neurophysiology and neuropharmacology of ad- 
diction 223 

Central nervous system depressants 223 

Psychological studies of addiction 225 

Conditioning factors 227 

Mental set 228 



Page 

Clinical investigations — NINDB 229 

Medical neurology 230 

Neurochemistry 230 

Biophysical applications 233 

Clinical neuropharmacology 235 

Neuroradiology 237 

Electroencephalography 241 

Clinical neurophysiology 241 

Surgical neurology 243 

Neuropathology 244 

Ophthalmology 251 

Summary 259 

National Institute of Allergy and Infectious 

Diseases 261 

Introduction 261 

Respiratory virus and enterovirus studies 263 

Definition of specific viral diseases 263 

Respiratory viral vaccines 264 

Seroepidemiologic studies 264 

Specific respiratory disease studies 264 

Respiratory viruses shared by man and domestic 

animals 265 

Studies of tumor (or cancer) viruses 265 

Studies of tumor viruses in nature 266 

Studies in medical mycology 267 

Biochemistry of antibiotics 267 

Streptococci and other human bacterial pathogens. . . 268 
Fractionation of [hydrogen isotopes by a marine 

pseudomonad 268 

Q fever studies at Rocky Mountain Laboratory 268 

Colorado tick fever 269 

Bat rabies 270 

Fractionation of cell walls 270 

Antigens from tubercle bacillus 270 

Studies of delayed hypersensitivity 271 

Purification of viruses 271 

Tick paralysis 271 

Germ-free animal research 272 

Germ-free mouse kidney tissue culture 272 

Middle- America research unit 273 

Histoplasmosis in Panama 273 

Arthropod-borne virus studies 274 

Bacterial diseases 274 

Malaria — Human 274 

Malaria — In monkeys 275 

Drugs — Mechanism of action 275 

Drugs — Pharmacology and Lachesiology (fate) 276 

Drugs — Trials against intestinal parasites 276 

Drugs and nutrition as related to schistosomiasis .... 276 

Helminthic infections in mice 276 

Molluscicides 277 

Metabolic changes in parasitic infections 277 

Toxoplasmosis 277 

Axenic cultivation 278 

Immunology 278 

Treatment of fungus infections 279 

Clinical studies of viral diseases 279 



XIV 



CONTENTS 



Page 

Staphylococcal problem 279 

Antibodies in ascitic fluid 280 

Filariasis 280 

Lupus erythematosus 280 

Biology of viruses 280 

Cell biology 281 

Biosynthesis of virus 281 

National Institute of Dental Research 283 

Introduction 283 

Laboratory research activities 286 

Clinical Research activities 290 



Page 

Division op Biologics Standards 293 

Introduction 293 

Laboratory of viral products 294 

Poliomyelitis vaccine 295 

Live poliovirus vaccine 295 

Measles vaccine 296 

Rickettsial vaccines 296 

Respiratory viruses 296 

Viruses — General 296 

Miscellaneous 297 

Laboratory of bacterial products 297 

Laboratory of blood and blood products 298 

Laboratory of control activities 299 



NATIONAL CANCER INSTITUTE 



INTRODUCTION 

An increasingly vigorous and productive re- 
search program prosecuted by the intramural staff 
of the National Cancer Institute during 1959 
makes this summary of selected accomplishments 
even less adequate than its predecessors. New 
approaches to critically important problems in 
the study of neoplastic diseases characterized the 
year's work. Group enterprises flourished to a 
greater extent than before, yet the talented indi- 
vidual who prefers to work largely by himself de- 
servedly received equal encouragement. Ideas 
are born in individual minds and the communica- 
tion of these ideas is probably the single most im- 
portant stimulus to productive scientific research. 
Thus, creation of an environment conducive to in- 
tellectual give-and-take becomes management's 
most important function. There is no single or 
easy way to achieve this environment in a large 
organization which depends for its vitality on in- 
dividual initiative and intellectual curiosity. 

The virus oncology program, for example, com- 
mands respect for its consistently superior per- 
formance. Part of its effort is centered in person- 
nel of the Virus Oncology Section of the Labora- 
tory of Biology, but equally important are in- 
dividuals in several different laboratories who are 
interested in developing significant segments of 
the total program by their own ingenuity and have 
only intermittent contacts with colleagues of 
similar interests. 

The entire area of chemotherapeutic research, 
much larger at this point than the virus program, 
has been more strongly influenced by a few 
acknowledged leaders. While this area is com- 
partmentalized to a degree, there is a greater ten- 
dency for the individual investigators to exchange 
information on a continuing basis. 

These examples, which could be extended, 
simply indicate that research is people and that 
people differ in many important ways. The or- 
ganizational chart, so dear to the heart of the pro- 
fessional administrator, can indicate channels for 
conducting ordinary business but can also stifle 



creative endeavor if taken as a blueprint for the 
conduct of research. Clearances, confirmations, 
and authorizations required by a mammoth Fed- 
eral establishment can lead to personal frustra- 
tion, impede progress, and cause the affected to 
seek employment elsewhere. Substantial decen- 
tralization of decision-making, through the whole- 
hearted cooperation of the Laboratory and 
Branch chiefs and other senior staff members, has 
helped reduce a number of these petty but highly 
sensitive problems. The roles of the Administra- 
tive Officer and Administrative Assistant have 
been especially valuable in reducing causes of 
friction ; yet much remains to be done. 

Since the size of the working group centered 
about a scientific leader can be determined by the 
leader's ambitions, personality, and research com- 
petence, the section becomes the key part of the 
intramural organization. The long-range plan- 
ning and business aspects of the operation require 
an individual actively engaged in a personal re- 
search program but able and willing to accept even 
broader responsibility — the laboratory and branch 
chief. The last few years have seen increasing 
concentration behind those with leadership capa- 
bilities. The head of a section has generally fared 
well. Some laboratory or branch chiefs, on the 
other hand, have been so overburdened with so 
many different responsibilities that their personal 
research, from which they derive a large degree of 
satisfaction, has been subordinated to the common 
good. It seems desirable, therefore, to reduce the 
size of some organizational segments of the Insti- 
tute by creating one or more new laboratories or 
branches, probably during 1960, rather than to 
persist in stultifying research activity through 
imposition of related but essentially noncontribu- 
tory duties on the Institute's leaders. 

The death of Dr. Jesse Philip Greenstein, first 
Chief, Laboratory of Biochemistry, on February 
12, closed the career of one of our most distin- 
guished, imaginative, and productive scientists, 
a beloved friend and counselor, and a world leader 
in biochemistry and cancer research. Dr. Green- 
stein worked at a furious pace in 1939 when he 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



joined the group that became the National Cancer 
Institute, and that pace never slackened. His 
interests were extremely broad. His mature judg- 
ment, boundless energy, lucidity of expression, 
and his firm though understanding, even jovial, 
nature made a lasting impression on even the 
most casual acquaintance. He was completely 
loyal, and he had a profound effect on the devel- 
opment of the Institute's research program, which 
often extended into many areas of the Institute's 
expanding activities. Very few people realized 
the intense and benevolent interest he showed in 
the personal problems of his associates. Dr. 
Greenstein contributed most importantly to areas 
of amino-acid, peptide, and protein biochemistry, 
including enzymology. He defined the concept of 
anaplasia in biochemical terms, and left behind a 
monument, Biochemistry of Cancer, one of the 
most quoted works in the enormous literature of 
cancer. 

Dr. Herbert A. Sober has succeeded to the posi- 
tion, Chief, Laboratory of Biochemistry. 

Dr. Delbert Mauritz Bergenstal, Assistant 
Chief, Endocrinology Branch, died on September 
12 after a long illness. A close associate of Dr. 
Charles Huggins, Dr. Bergenstal joined the staff 
of the National Cancer Institute in 1955 after a 
brilliant career at the University of Chicago. He 
was a superb clinician with great ability in 
clinical investigation. His warm magnetic person- 
ality, his tremendous drive, and infinite patience 
endeared him to his colleagues and his patients. 
His arguments were imaginative, his exposition 
stimulating. Among his important contributions 
to the endocrinology of cancer are studies on the 
metabolic influences of ACTH, cortisone and 
growth hormone, characterization of catabolic re- 
sponses induced by adrenal and thyroid hormones, 
the effect of adrenalectomy on advanced prostatic 
and mammary cancers, and the effect of DDD on 
adrenal cancer. 

Dr. Mortimer B. Lipsett has been appointed 
Assistant Chief, Endocrinology Branch. 

Dr. Nathaniel I. Berlin was named Chief, Gen- 
eral Medicine Branch, a duty most capably dis- 
charged for 5 years by Dr. Charles G. Zubrod, in 
addition to his many complex responsibilities as 
Clinical Director. 

Two meetings of the Board of Scientific Coun- 
selors were held during 1959. Drs. Charles Hug- 
gins, Carl V. Moore, and Eugene P. Pendergrass 



retired on June 30 from the Board on completion 
of their appointments. Such events, though neces- 
sary, cause deep regret, especially when the rela- 
tionships between the staff and the Board are as 
cordial as were those with the original Scientific 
Counselors. Continuing excellent relationships 
are in prospect, since the new members, Drs. Hugh 
E. Butt, J. Engelbert Dunphy, and Jacob Furth, 
are just as deeply interested, understanding, and 
cooperative as were their predecessors. Dr. Wen- 
dell M. Stanley relinquished his position as Chair- 
man of the Board but consented to remain in active 
membership through June 30, 1961, with Drs. 
E. K. Marshall and Philip P. Cohen. Dr. Cohen 
is the new chairman. 

The Board of Scientific Counselors continues to 
express confidence in the research staff and its 
program. Their discussions are most helpful. 
Differences of opinion, characteristic of a good 
and intimate working relationship, are resolved by 
free and frank discussion to everybody's ultimate 
satisfaction. The meetings to date have required 
unduly intense effort from the counselors, who 
meet throughout the day with various members of 
the staff and discuss program among themselves 
far into the night. Different types of meetings 
are being discussed with the chairman as a means 
of reducing the workload. 

The increasing number of scientific meetings 
demanding the participation or attention of the 
staff, although gratifying in the sense of con- 
ferring some distinction solely for research ac- 
complishment, is also disturbing because so much 
of our talented associates' efforts must be devoted 
to preparing papers. Program has not been seri- 
ously damaged as yet, but the saturation point 
has probably been reached. Foreign travel, 
though not yet as commonplace as domestic, has 
become the order of the day. The problem is al- 
most impossible to control as long as the upper 
echelons of government are international-minded, 
and in some cases actually solicit the participation 
of staff members in planning foreign ventures. 
Every request for travel to foreign meetings has 
had obvious merit in relation to research, and the 
ability and desire of extramural organizations and 
groups to finance travel for National Cancer In- 
stitute staff have increased materially. 

Tangible recognitions of leadership in cancer 
research on both national and international bases 
have been accorded to many persons in intramural 



NATIONAL CANCER INSTITUTE 



research. Dr. W. C. Hueper received the 
A.A.A.S-Anne Frankel Rosenthal Award for 1959 
in recognition of his contributions to knowledge 
of the causation of cancer in man. Dr. Harold L. 
Stewart retired as President of the American As- 
sociation for Cancer Research. Dr. Murray J. 
Shear is the Vice President of the Association, and 
Dr. Thelma B. Dunn was elected to the Board of 
Directors. Dr. Dunn is also President of the 
Washington Society of Pathologists. Dr. Roy 
Hertz was appointed to the Research Advisory 
Council of the American Cancer Society for a five- 
year term. Dr. L. W. Law has been named as 
delegate to the World Health Organization's Ad- 
visory Committee on Cancer. Dr. A. J. Dalton 
is President of the New York Society of Electron 
Microscopists. 

An exhibit, "Tumor Cells in Blood," by Drs. 
J. F. Potter, R. F. Kaiser, R. A. Malmgren, A. W. 
Hilberg, and J. C. Pruitt received an Award of 
Merit at the annual meeting of the American 
Medical Association. 

Causes of Cancer 

Twenty years of experience in carcinogenesis re- 
search was highlighted late in the year through 
governmental actions required by amendments to 
the Federal Food, Drug, and Cosmetic Act en- 
acted in 1958. Several Institute staff members 
were most helpful in furnishing advice to inter- 
ested parties on general and specific aspects of 
carcinogenesis. A meeting of some 25 investiga- 
tors experienced in research on cancer causation 
was held during December to determine the extent 
to which agreement could be reached on different 
facets of the complex carcinogenesis picture. A 
lively discussion elicited these important points: 

1. Carcinogenic property is not comparable to a 
physical or chemical property. 

2. Groups of compounds possessing common 
biological properties may be carcinogenic by vir- 
tue of those properties, specifically : ionizing radia- 
tions, estrogens, and goitrogens of the thiourea 
type. 

3. Determination of carcinogenic property re- 
quires judgment as to the validity of the experi- 
mental design and its adequacy in relation to the 
known qualities of a compound and its relatives, 
to biological availability, to the reasonable inter- 
pretation of results and to the reproducibility of 



the responses. Many isolated experiments re- 
corded in the older literature claiming carcino- 
genic properties for certain chemicals remain un- 
substantiated and are of questionable validity. 

4. The potential hazard of a chemical com- 
pound can be estimated only on the basis of cur- 
rently known facts and each situation must be 
judged on its own merit. 

Much more remains to be learned about carcino- 
genesis, and, indeed, substantial progress is being 
made in both practical and theoretical aspects. 
We conceive our duty to be the accumulation of 
additional facts and especially basic principles 
which may be applied broadly to general and spe- 
cial problems, rather than to embrace an extensive 
testing program of large numbers of compounds 
for purely pragmatic purposes. At the same time, 
however, we are glad within the limits of our 
knowledge to advise others interested in such 
testing, which may well assume increasing im- 
portance. 

CHEMICALS 

The ability of some viruses to induce cancers in 
mice only when injected shortly after birth has 
raised a question as to the relative sensitivity of 
infant and adult mice to the action of chemical 
carcinogens. Information advanced by Shubik 
suggests a heightened carcinogenic response to 
9 : 10 dimethyl-1,2 benzanthracene. Drs. Margaret 
Kelly and R. W. O'Gara have injected microgram 
quantities of 3-methylcholanthrene or dibenz[a,h]- 
anthracene subcutaneously into newborn Swiss 
mice and mice of strain C3H. Both respond by 
increased frequency in pulmonary tumors of the 
characteristic murine type recognizable as early 
as 16 weeks and becoming progressively more nu- 
merous thereafter. The incidence of subcutaneous 
sarcomas, however, does not keep pace with that 
of the pulmonary neoplasms. The same procedure 
elicits fewer tumors of the lung in mice of strain 
C5YB1. Other types of cancer have appeared 
sporadically. This research is being extended to 
other chemical carcinogens, since the differences 
already observed suggest both qualitative and 
quantitative importance. 

Drs. W. C. Hueper and W. W. Payne have been 
cooperating with Messrs. A. C. Stern and E. C. 
Tabor (Taft Sanitary Engineering Center) and 
with Dr. Paul Kotin (University of Southern 



4 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



California) in a study of cancer production in 
mice by dusts collected from several American 
cities. The benz[a]pyrene content of each sample 
has been analyzed so that the cancerous response, 
which can confidently be expected from the ex- 
perience of Drs. Joseph Leiter, M. B. Shimkin, 
and M. J. Shear reported in 1942, may be related 
to a reference standard. Aliquots of each sample 
have also been divided into several fractions repre- 
senting different types of aromatic or aliphatic 
compounds. Preliminary results indicate differ- 
ences in the carcinogenic potency of the fractions 
or extracts relating both to the source of the start- 
ing material and to the chemical nature of the 
fraction tested. In at least two cases, the aliphatic 
fractions appear to be more carcinogenic than 
those composed of aromatic compounds. 

Further experience with certain chromium com- 
pounds now permits Dr. Hueper to evaluate their 
relative carcinogenicity for rats, when injected 
intramuscularly or intrapleurally in this descend- 
ing order: calcium chromate, sintered calcium 
chromate, sintered chromium trioxide, zinc chro- 
mate, strontium chromate, barium chromate 
(weak), and lead chromate (noncarcinogenic). 
Dr. Payne reports calcium chromate and sintered 
calcium chromate carcinogenic for mice when 
sheep fat is used as the vehicle for subcutaneous 
injection. 

Fractionation by particle size and chemical 
analysis of the residue from leached chromite ore 
reveal that the smaller particles contain higher 
proportions of hexavalent chromium, thus in- 
creasing their acute toxicity when inhaled. Re- 
moval of water soluble compounds from the resi- 
due does not alter the degree of carcinogenic po- 
tency, according to Drs. Payne and Hueper. 
Evidently both the discarded part of the roast as 
well as the desired mixture of chromium com- 
pounds may be carcinogenic hazards in industry. 

Earlier reports have recorded the production of 
neoplasms by subcutaneous injection of a variety 
of water insoluble polymers studied by Dr. 
Hueper. He now has completed a study of water 
insoluble polyvinyl pyrrolidones, polyvinyl alco- 
hol, and dextrans. Sarcomas develop in those 
organs in which the injected material is deposited, 
especially in the reticuloendothelial system. 
Polymers of the same type, but produced by differ- 
ent processes, possess different carcinogenic poten- 



cies and neither chemical nor physical factors of 
the molecules themselves can be recognized to ac- 
count for the differences. These results seem al- 
most identical with those obtained with water- 
soluble compounds of the same general classes. 
The deposits are easily recognized in the tissues 
into which they were originally introduced or sub- 
sequently transported. Possibly the physical state 
within the cells may be partially responsible for 
the carcinogenic effect. 

Silicone gum and powdered silica are inert, 
but silicone rubber, introduced into the sub- 
cutaneous tissues of rats, produces neoplasms 
when the molecule contains many cross linkages. 
Linear molecules of silicone rubber are weakly 
carcinogenic at best. Polyethylene is moderately 
carcinogenic when implanted subcutaneously as 
films or discs but is inactive as a powder, a prin- 
ciple established earlier by the Oppenheimers for 
other plastics. Polyurethane as a sponge also 
produces connective-tissue tumors. 

Heated fats and charred foods are known to 
contain substances that can elicit a cancerous 
response in mice. Some years ago Dr. Hueper 
became interested in the potential carcinogenic 
hazard from coffee consumption. After Dr. M. 
Kuratsune identified benz[a]pyrene in coffee soots, 
a limited study was initiated with the cooperation 
of various members of the coffee industry. Drs. 
Kuratsune and Hueper now conclude that aro- 
matic hydrocarbons are present in most roasted 
coffee beans in extremely low concentration. Only 
certain "dark roasts," which form an extremely 
small fraction of the total production of coffee 
and are characteristically made by family con- 
cerns, contain any detectable amount of known 
carcinogenic hydrocarbons. Coffee consumption, 
then, plays no significant role in human carcino- 
genesis. 

Soots from coffee-roasting plants are an im- 
portant commercial source of caffeine, in which 
benz[a]pyrene is quite soluble. Drs. Kuratsune 
and Hueper made use of this fact to study distri- 
bution of the carcinogen in the stomach of the 
mouse. Intense fluorescence develops in the 
rumen of the stomach, where benz[a]pyrene is 
known to elicit tumors when fed, but does not 
appear in the glandular mucosa in which benz[a]- 
pyrene evokes no cancerous response. Earlier 
experiments reported by Dr. H. L. Stewart have 



NATIONAL CANCER INSTITUTE 



clearly shown that some carcinogenic hydrocar- 
bons elicit some adenocarcinomas when injected 
directly into the glandular gastric mucosa. The 
entire experience reemphasizes the need for study 
of gastric secretions in relation to cancerigenesis 
in the stomach. 

Study of causation of gastric cancer has been 
hampered by lack of reliable and reproducible 
methods for producing adenocarcinomas of the 
stomach. There has been some progress following 
the discovery by Drs. H. P. Morris and H. L. 
Stewart of a few lesions in the glandular stomach 
of rats fed 2,7 fluorenylenebisacetamide. The 
same lesions seen in human material would unques- 
tionably be diagnosed as adenocarcinomas, but ex- 
perience over the years has revealed a number of 
gastric lesions in mice that simulate gastric cancer 
but have proved to be non-neoplastic. It is essen- 
tial, therefore, to have a clear picture of the bio- 
logical behavior of gastric lesions when interpret- 
ing significance of morphological changes. While 
some of the lesions have invaded the muscularis 
in the rats fed 2,7 fluorenylenebisacetamide, no 
metastases have been recognized. All of the sub- 
jects develop metastasizing carcinomas of the 
small intestine, and some also have malignant 
tumors of other sites including the salivary glands. 

Dr. W. D. Conway and Miss E. J. Lethco have 
examined the purity of several commercial prepa- 
rations of the dyes yellow AB and yellow OB. 
The Food and Drug Administration limits the 
quantity of /3-naphthylamine in those products to 
500 parts per million and only 1 of the 10 samples 
contained more than the permissible quantity. 

Four melanomas have appeared among 51 
guinea pigs painted with 9,10-dimethyl-l,2 benz- 
anthracene by Dr. J. H. Edgcomb. 

Drs. M. L. Hesselbach (NIAID) and E. W. 
O'Gara report production of subcutaneous fibro- 
sarcomas in rats by multiple injections of fast 
green or light green. The earliest tumors appear 
at 7 months. The sarcomas are readily transplant- 
able and frequently metastasize. 

MECHANISMS OF CARCINOGENESIS 

Study of the mechanism by which N-2-fluorenyl- 
acetamide produces cancers in rats has required 
identification of excretion products, their syn- 
thesis and bioassay, chemical modification of the 
parent molecule, and evaluation of carcinogenic 



potency of the new compounds. Much has been 
learned from this approach and several new car- 
cinogens with interesting types of activity have 
been described, such as 2,7-fluorenylenebisacet- 
amide. The experience to date indicates that hy- 
droxylation of N-2-fluorenylacetamicle in posi- 
tions 1, 3, 5, or 7 reduces its carcinogenic potency, 
which Dr. Morris interprets as a detoxification 
mechanism or a means of solubilization to facili- 
tate excretion. Fluorination in the omega position 
of the acetyl group enhances carcinogenic potency, 
and two fluorinated compounds produce intestinal 
tumors when fed to rats, but hepatomas when fed 
to dogs. 

The influence of dietary factors on N-2-fluo- 
renylacetamide has also been studied by Dr. 
Morris. A high dietary intake of pyridoxine re- 
sults in many mammary tumors among rats eating 
N-2-fluorenylacetamide. If the mammary lesions 
are removed surgically, the rats ultimately develop 
hepatic neoplasms. Conversely, rats with low die- 
tary intakes of vitamin B6 develop tumors of the 
liver but not of the breast. 

Detailed studies by Dr. H. M. Dyer of the me- 
tabolism of N-2-fluorenylacetamide in Buffalo rats 
which ate a tryptophan-rich diet were recorded 
in last year's report. The objective (as reported 
by Dunning) had been the production of tumors 
of the urinary bladder; yet none occurred. The 
same procedure does produce cancers of the blad- 
der in Fischer rats, however, and Dr. Dyer reports 
no chemical differences in the excretion products 
of N-2-fluorenylacetamide or of tryptophan in the 
two strains. Addition of tryptophan is not essen- 
tial to cancer production. Once again, the genetic 
constitution of the host becomes a most important 
factor in carcinogenesis. 

Dr. Dyer finds differences in tryptophan me- 
tabolism in rats fed hepatic carcinogens and chem- 
ically related but noncarcinogenic compounds ; she 
ascribes this to differences in the extent of dam- 
age produced in the liver. 

Greater attention is now being directed towards 
the fate of N-2-fluorenylacetamide in organs and 
tissues where the compound is bound to proteins. 
The Doctors Weisburger report that binding of 
N-2-fluorenylacetamide labeled with C 14 cannot be 
explained on the basis of artifacts created by in 
vitro manipulations. Furthermore, orthohydrox- 
ylated compounds form stable complexes with a 
variety of metal ions. Introduction of a hydroxyl 



6 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



group at a greater distance in the molecule does 
not confer the same property. 

Dr. H. F. Blum, reviewing his vast experience 
in the quantitative study of ultraviolet carcino- 
genesis, writes in his most recent book : 

"... By treating the quantitative description 
as empirical, one may arrive at certain conclusions 
regarding this kind of carcinogenesis. 

"It seems clear that whatever the process of 
cancerization, it is continuous throughout the 
whole of the development of the cancer and cannot 
be separated into distinct periods. 

"Cancerization is in some way cumulative — if 
dosage is stopped, development continues at a re- 
tarded pace but speeds up with renewal of dosage. 

"Although there is evidence of a small degree of 
recovery, this is slight in overall effect, and car- 
cinogenesis may be regarded as essentially irre- 
versible. 

"Since there is recovery — and we take particular 
account of the evidence of this in the failure of 
reciprocity at very low close-rates — it seems neces- 
sary to conclude that there is a threshold dosage 
at which recovery just balances cancerization. 
But this threshold is certainly very low and is not 
directly measurable because of the limited lifetime 
of the animals. Thus, in any practical sense, 
carcinogenesis by ultraviolet light may be con- 
sidered as essentially nonthreshold." 

Dr. Blum has examined the possibility of similar 
cumulative effects among chemical carcinogens, 
but finds the quantitative data now available in- 
adequate to decide the point. 

With Mr. G. A. Soffen (Princeton University), 
Dr. Blum is studying the effects of single doses 
of ultraviolet irradiation on mouse skin. The 
original insult renders cells incapable of dividing, 
but five different doses result in normal mitotic 
counts at 1.5 days. A strong hyperplastic response 
follows in the epidermis with higher proliferation 
rates than are found in cancers resulting from re- 
peated doses of ultraviolet. The hyperplastic re- 
sponse falls off with time. The higher the dose, 
the greater the number of cells destroyed and the 
greater the initial rate of cell proliferation as 
measured by mitotic indices. 

VIRUSES 

The problem of identifying viruses that may 
cause cancers in man is extremely complex. One 



group of investigators and 3 individual scientists 
working with different techniques have screened 
more than 100 clinical cancers for viruses. Only 
one of these people has found a clearly identifiable 
virus. The recovery of a number of viruses from 
transplanted experimental tumors such as sarcoma 
37 is quite common and includes two new varieties 
of murine hepatitis virus found recently by Dr. 
R. A. Manaker in experimental leukemias. 
Either the cancers that affect man are basically 
different in their ability to harbor passenger 
viruses or we are working toward isolation with 
comparatively insensitive tools. Continuing re- 
evaluation of the techniques employed in the 
search for viruses in human cancers proceeds, to- 
gether with encouragement of individual initia- 
tive in modifying techniques of tissue extraction 
and tissue culture, in the belief that passenger 
viruses inhabit some proportion of cancers that 
affect man. 

Dr. S. E. Stewart has observed a peculiar reac- 
tion in monolayer cultures of freshly isolated hu- 
man amnion or embryonic cells produced by con- 
centrates of two different human cancers and the 
urines of three children with neoplastic diseases. 
Focal areas of increased growth appear in the 
affected cultures leading to the formation of dis- 
crete dense, raised patches, easily visible to the 
naked eye. Although serial passage in tissue cul- 
ture of the proliferative effect is readily accom- 
plished by use of supernatant fluids, the cell-free 
nature of such fluids has not been established. 
Filtration in bacteriaproof systems or centrifuga- 
tion at high speed destroys capacity of the fluid 
to transmit the proliferative effect. The sediment 
obtained from these procedures retains the ability 
to cause a focal proliferative response in new cul- 
tures, and this property is maintained after re- 
peated freezing at the temperature of dry ice and 
intermittent thawing. The nature of the agent 
responsible is not known. 

Injections of many tissue-culture preparations 
or tissue extracts into newborn hamsters and mice, 
and observations of the animals for long periods, 
have reemphasized the need for better informa- 
tion on diseases occurring naturally among labora- 
tory species. Attention has been drawn to a curi- 
ous but reasonably common lesion of the placenta 
in hamsters often resulting in fetal death in utero. 
Hamsters also develop ulcerating and/or polypoid 
lesions of the intestines which bleed profusely. 



NATIONAL CANCER INSTITUTE 



Polyoma virus or parotid tumor agent 

Some confusion may be caused by use of the 
synonyms polyoma virus and parotid tumor agent 
for an agent which produces multiple primary 
neoplasms in mice and tumors in hamsters, rats, 
and rabbits. 

The chemical nature of polyoma virus has been 
studied by scientists of the Sloan-Kettering In- 
stitute in collaboration with Drs. Stewart and 
Eddy. Infective nucleic acids prepared after the 
techniques of Gierer and Schramm or of Kirby 
are inactivated by deoxyribonuclease, but by ribo- 
nuclease as measured by effects on cytopathic 
changes in tissue culture, hemagglutination of 
guinea pig erythrocytes, and tumor induction in 
mice and hamsters. The infectivity of intact 
viruses is not influenced by either of the enzymes. 
The infective component of polyoma, therefore, 
would appear to be a deoxyribonucleic acid. 

Early in the year Dr. W. G. Banfield demon- 
strated intranuclear, intracytoplasmic, and ex- 
tracellular, relatively uniform, spherical particles 
27 to 35 millimicrons in diameter in lymphoma 
cells grown in tissue culture by Dr. C. J. Dawe 
for propagation of the parotid tumor agent. Un- 
inoculated cultures contained no similar bodies. 
The descriptions provided by Drs. Banfield and 
Dawe are comparable to the measurements re- 
ported last year by Dr. H. Kahler and colleagues 
on isolated particles possessing polyoma activity 
when allowance is made for the different methods 
of preparation. Drs. Banfield and Dawe consider 
that propagation of the virus is predominantly 
intranuclear. The intranuclear localization of 
polyoma demonstrated by fluorescent antibody 
techniques in tissue cultures of a lymphoma, as 
reported by Drs. R. A. Malmgren, A. S. Rabson, 
and Giancarlo Rabotti (Visiting Scientist), sup- 
ports that view. These workers, however, have 
not been able to demonstrate polyoma virus in 
parotid-gland tumors by fluorescent antibody 
techniques. Others have shown that not all 
tumors produced by polyoma contain detectable 
amounts of the virus when tissue-culture methods 
are employed. 

Dr. Robert Love has applied his sensitive tolui- 
dine blue molybdate methods for the demonstra- 
tion of intracellular ribonucleoproteins to the 
study of lymphoma cells infected in vitro with 



polyoma. Neither nuclear parachromatin nor the 
nucleolinus can be demonstrated. The nucleolus 
is enlarged, and vacuoles containing a diffuse and 
condensed, abnormally staining, form of ribo- 
nucleic acid appear in the nucleus. The nucleus 
first enlarges, then shrinks and eventually disinte- 
grates by karyorrhexis. Dr. Love has not been 
able to identify a deoxyribonucleic acid in associa- 
tion with the newly formed intranuclear ribo- 
nucleoprotein by histochemical procedures. 

Dr. Rabson and Dr. Ruth Kirschtein (DBS) 
have produced intracranial sarcomas through the 
intracerebral inoculation of polyoma virus in ham- 
sters. These neoplasms appear to arise from the 
pia mater and the adventitia of the meningeal and 
cerebral blood vessels. 

Drs. Stanton, Stewart, and Eddy report that 
injection of polyoma virus into Swiss mouse 
fetuses in utero late in fetal life results in earlier 
death and a higher incidence of tumors than injec- 
tion on the first day of extrauterine life. Mice in- 
fected as fetuses develop no new histological types 
of neoplasms. Some of the mice, however, born 
to mothers reared in close proximity to adult mice 
known to harbor parotid-tumor agent had a signi- 
ficantly lower incidence of salivary-gland tumors 
and "renal tubular lesions." A comprehensive 
analysis of histological changes by Dr. Stanton 
reveals that some cells in the infected mice hyper- 
trophy, form intranuclear inclusions, and degen- 
erate. The neoplasms in the series are composed of 
hyperplastic epithelial elements and proliferating 
or differentiated cells, limited in their capacity to 
invade surrounding tissues or to metastasize. 
Some tumors regress; others kill their hosts by 
local growth in vital organs. 

In another study of polyoma virus infection, 
Dr. Sarah Stewart, Dr. Kahler, and Dr. Stanton 
have studied the tumors occurring with limiting 
dilutions of virus culture preparations subjected 
to differential high-speed centrifugation. All 
fractions produce tumors, but the greatest activity 
is found in sediments produced with forces de- 
signed to contain the greatest quantity of the 
agent. Again, the variety of neoplasms produced 
by the fractions is similar. Analysis of the tissue 
reactions that follow minimal doses after long- 
term study reveals many nonspecific lesions in 
older mice, especially in the kidneys and salivary 
glands, resembling either abortive attempts at cell 



8 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



proliferation or regression of minimal lesions. 
Older mice do develop a significant number of 
neoplasms, particularly bone tumors that show 
clear evidence of the capacity to invade and 
metastasize. Dr. Stanton comments: 

"The polyoma virus-tumors serve as exceptional 
experimental tools. The wide range of species 
that respond and the convenience in working with 
small rodents constitute an advantage over chemi- 
cally induced tumors both in the short, latent peri- 
od and opportunity of following the progress and 
behavior of the tumors in relation to virus as de- 
tected by new virological and morphological tech- 
niques . . . There is good evidence to indicate 
that induction and progression proceed in a slow, 
stepwise fashion which may allow separate study 
of the two most important attributes of tumors, 
namely, cell proliferation and the acquisition of 
aggressive behavior patterns by the proliferating 
cells." 

Perhaps the most significant part of this state- 
ment is the recognition of phases in the evolution 
of the cancerous state among neoplasms not asso- 
ciated with known endocrine functions. 

Especially important to the search for viruses 
that may cause cancer in man is the ability to 
recognize a cancerous change in some foreign 
species or semiartificial system such as tissue cul- 
ture. Last year's report referred to the most in- 
teresting changes produced by parotid-tumor 
agent in salivary gland anlagen grown in vitro by 
Dr. Dawe. He now reports that increasing 
chronological age to as much as 15 months does 
not diminish the proliferative response of submax- 
illary glands of mice to polyoma virus in tissue 
culture. If anything, the reaction, which mimics 
neoplasia in its morphological characteristics, is 
more pronounced. The altered cells still fail to 
produce local neoplasms when transplanted to 
genetically appropriate hosts, although evidence 
for virus transfer is clear. On the other hand, in- 
fection in vitro of salivary gland rudiments in the 
earliest stages of morphogenesis produces little in 
the way of a neoplastic response. 

Dr. Dawe also reports that growth of adult 
parotid gland in sponge matrix culture results in 
digestion of the sponge by a protease secreted by 
the cells. 

While Dr. K. K. Sanford has been able to prop- 
agate polyoma virus in a clone from normal C3H 



mouse parotid tissue and in a long-term strain 
of C3H embryonic fibroblasts, two strains of C3H 
adult fibroblasts and one strain of Chinese hamster 
cells, although supporting limited maintenance, 
show no conspicuous effects of infections. 

The failure of certain newborn mice to develop 
tumors when injected with polyoma virus may, 
in part, be due to the presence of antipolyoma 
antibodies in mothers' milk transmitted to the 
nurslings. Antibody titers as measured by in- 
hibition of hemagglutination are as high in milk 
as in sera, according to Drs. Stewart and Eddy. 
Similar reasoning would account for the failure 
of naturally infected stocks of mice to develop 
salivary-gland tumors so characteristic of 
polyoma-virus action. Dr. L. W. Law reports 
that mothers whose blood contained hemaggluti- 
nation inhibiting antibodies in titers greater than 
200 delivered young which resisted the oncogenic 
action of the parotid-tumor agent. Foster nurs- 
ing experiments conducted by Dr. Law point to 
the transfer of antibodies in the milk of infected 
mothers and also suggest that antibodies may be 
transmitted transplacentally. 

Leukemia virus (Moloney) 

The murine leukemia virus isolated by Dr. J. B. 
Moloney from transplantable sarcoma 37 is being 
studied intensively. Standardized techniques 
based on differential centrifugation at alternating 
high and low speeds or on bacteriological filtra- 
tions combined with differential ultracentrifuga- 
tion yield equally potent preparations from sar- 
coma 37 or from transplantable leukemias pro- 
duced by the virus. Leukemic spleens or lymph 
nodes are excellent sources of the agent, but the 
liver is a relatively poor one. 

BALB/c strain mice have been used by Dr. 
Moloney as a reference standard in studies of viral 
leukemogenesis. Susceptibility is retained for at 
least 5 months, but the latent period is somewhat 
longer among older mice. Mice of strains C3H, 
C3Hf, A/LN, I, Kill, DBA/2, C57BL and ran- 
domly bred Swiss mice all develop leukemia from 
virus infections. The incidence approximates 100 
percent and the latent intervals of leukemia pro- 
duction do not vary significantly except for 
strain C57/BL, which develops the disease in only 
about 30 percent of infected subjects. The E* 



NATIONAL CANCER INSTITUTE 



9 



hybrids obtained by mating each of the strains 
noted above with BALB/c mice respond to leu- 
kemogenesis as promptly as do mice of the refer- 
ence strain. 

Potency of the virus has been increased by selec- 
tive virus passage in vivo. After eight passages 
the latent interval has decreased from 6.4 months 
to 10-12 weeks in all mice tested. 

Dr. T. B. Dunn reports that this leukemia is 
morphologically indistinguishable from spontane- 
ously occurring lymphocytic leukemias in several 
mouse strains. The lymph nodes, spleen, liver, 
and thymus are characteristically enlarged and 
infiltrated with typically lymphoid cells that also 
involve the salivary glands, kidneys, lungs, and 
meninges in the advanced stage of the disease. 
The same histologic pattern is seen in all mice 
examined whether the leukemia is transmitted by 
cell-free preparations or by leukemic cells. Blood 
from the leukemic mice presents moderate leuco- 
cytosis and anemia. No neoplasms other than 
those of the lymphocytic type have as yet been 
observed in any of the test animals. Leukemia 
can be produced by Dr. Moloney's virus by subcu- 
taneous, intravenous, intracerebral, or intraperi- 
toneal inoculation. 

The leukemia virus remains stable at tempera- 
tures below —50° C. for long periods of time when 
prepared in citrate buffer and withstands lyo- 
philization without apparent loss of activity. 
Biological activity is retained when the virus is 
exposed to 37° C. for 90 minutes but is lost at ex- 
posure to 56° C. within 30 minutes. 

Dr. A. J. Dalton has examined leukemias, pro- 
duced by Dr. Moloney's virus, under the electron 
microscope. Particles representing the agent be- 
long to morphological class Type A (Bernhard), 
have an external diameter averaging 100 milli- 
microns, and contain an electron dense nucleoid of 
48 millimicrons. They are not particularly abun- 
dant, but may be found in spleens, lymph nodes, 
and thymuses of infected mice and are found most 
frequently in the intercellular spaces and only oc- 
casionally within intracytoplasmic vacuoles. 

The Moloney virus is difficult to propagate in 
tissue culture, Dr. R. A. Manaker presents evi- 
dence that the agent can be maintained in vitro, 
but its behavior is not entirely clear at this time. 
Ameboid cells found in cultures of liver, thymus, 
spleen, or lymph nodes of leukemic mice seem to 



maintain the virus best, while cells clearly recog- 
nizable as lymphomatous support it little, if at all. 
Although many other types of cells have been used 
to propagate leukemia virus, activity has usually 
died out within four or five serial passages in vitro. 
Preliminary evidence obtained by Dr. Moloney 
suggests that immune sera prepared from rabbits 
receiving multiple injections of the leukemia virus 
do contain neutralizing antibodies. A diligent 
search by Dr. M. A. Fink for other antigenic 
properties which might be used in epidemiological 
studies has been unsuccessful. The agent does not 
fix complement, nor does it inhibit hemaggluti- 
nation. As a matter of fact, most of the oncogenic 
viruses are rather weakly antigenic. Polyoma is 
an exception. 

Mammary tumor agent 

Dr. H. B. Andervont recently described the dis- 
appearance of the mammary tumor agent from two 
female mice of strain Bill. Experiments de- 
signed to analyze the respective roles of the virus 
and the host in this phenomenon have included 
production of both C3H and Bill mice known 
respectively to carry and to be free of the agent. 
These stocks have been interbred. Ninety-five per- 
cent of the agent- free C3H mice exposed to their 
own mammary tumor inciter develop tumors of 
the breast. Breeding decreases the latent interval 
but has no effect on the incidence. Bill milk in 
C3Hf mice produces a smaller number of mam- 
mary carcinomas but is transmitted serially 
through the strain. It is already apparent, how- 
ever, that some RIII mice resist the carcinogenic 
effects of the C3H agent. "While the Bill agent 
is much less potent than the C3H mammary tumor 
inciter, the constitution of the host is also im- 
portant in determining carcinogenesis. Dr. An- 
dervont's earlier observations on the enhanced ac- 
tivity of the mammary-tumor agent obtained from 
wild mice and propagated through 20 generations 
in BALB/C, coupled with his other studies on the 
disappearance of C3H agent from mice of strains 
C57B1 and I, take on added significance. 

Further work along these lines has been con- 
ducted by Dr. W. E. Heston in his studies on genie 
influences associated with the propagation of the 
mammary tumor inciter. Segregation data ob- 
tained from the matings of strains C3H and 
C57B1 indicate that the agent can be propagated 



10 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



in the presence of one or several genes, rather than 
being under the control of a single gene. A female 
mouse lacking any of the genes required to propa- 
gate the agent does not eliminate it, but the virus 
does not multiply and is lost in subsequent genera- 
tions. The quality and quantity of mammary- 
tumor agent vary then according to the number 
of genes required for its propagation and their 
availability to the individual concerned. 

Dr. Sanford, with Drs. Dunn and Andervont, 
reports the persistence of mammary tumor inciter 
in cultures of certain cells for more than a year. 
Activity is lost if the cells take on a sarcomatous 
appearance. The usual structural characteristics 
of mouse mammary carcinomas are maintained 
when the cells grow in serum-supplemented, chem- 
ically defined, media but are lost when the me- 
dium contains serum and chick embryo extract. 

Rous sarcoma virus 

Examination of physical, chemical, and immu- 
nologic properties of the Rous sarcoma virus re- 
quires a highly potent source of material. Dr. W. 
R. Byran reports a four-fold increase in activity 
during the past year in viral preparations ob- 
tained by selective serial passage. This represents 
an increase of 100 times the potency of the best 
material available two years ago and makes puri- 
fication studies practical. The intranuclear and 
intracytoplasmic location of Rous virus antigen 
in Rous sarcomas has been demonstrated by Drs. 
Malmgren and Fink using fluorescent antibody 
techniques. 

Through the cooperation of Dr. J. A. Reyniers 
(Germ-Free Life Research Center, Tampa, Fla.), 
Dr. Bryan has acquired a number of Japanese 
quail (Coturnix japonica) for use in Rous virus 
research. These birds prove to be as susceptible 
to tumor production as the most sensitive chickens 
yet investigated. Yields of virus are fully as high 
from quail tumors as from chicken tumors. The 
small space required to house the Japanese quail, 
which are no larger than 5-day-old chicks, is a 
considerable advantage. 

OTHER CARCINOGENS 

While much is known about the quality of car- 
cinogenic responses to ionizing radiations, much 
remains to be learned concerning the mechanism 



of their action which can be approached through 
manipulations that increase or reduce the fre- 
quency of the response. Dr. Law, particularly 
interested in X-ray leukemogenesis, described a 
reduction in the incidence of X-ray-induced lym- 
phocytic leukemias in thymectomized C57BL mice 
some years ago. The normal response could be 
restored by subcutaneous transplantation of thy- 
mic fragments in which the leukemias apparently 
originated. Last year's report referred to the 
failure of thymic tissue placed intraperitoneally 
within millipore diffusion chambers to restore the 
capacity of thymectomized mice to develop leu- 
kemias after X-irradiation. The thymic tissue 
within the chamber does not become leukemic in 
either C57BL mice or in hybrids produced by 
mating C57BL to strain A. Nevertheless, 20 to 
30 percent of these subjects develop reticular 
neoplasms of Dr. Dunn's type A and B. No 
plasma cell neoplasms have been found under these 
conditions. 

Dr. Law now reports the prevention of neo- 
plastic changes in thymectomized mice bearing 
subcutaneous thymic grafts when isologous bone 
marrow is given immediately following the last of 
four weekly doses of total body X-irradiation. 
The incidence of reticular neoplasms other than 
lymphocytic leukemia is not affected. Chimeras 
produced by injection of AKR marrow into ir- 
radiated C3Hf mice, of Law or Bittner subline, 
less than 12 hours after birth develop the same 
incidence of leukemias as do the controls, nor is 
the leukemia incidence influenced by transplanta- 
tion of AKR thymic tissue. A most interesting 
observation, however, is a frequency of unilateral 
parotid tumors, approximating 10 percent among 
C3H/Bi mice receiving tissue from AKR donors 
known to be infected with polyoma virus as dem- 
onstrated by HI antibodies. 

Miss D. E. Uphoff and Dr. Law have extended 
their attempts at altering the high incidence of 
leukemia in irradiated AKR mice to include the 
use of marrow from four strains that are com- 
patible at the H-2 k locus. The results are incom- 
plete, but most of the leukemias arising to date in 
mice irradiated and protected when young have 
proved to originate in donor cells even though 
they come from strains with low frequency of 
spontaneous leukemia, Marrow treatment in AKR 



NATIONAL CANCER INSTITUTE 



11 



mice irradiated at six months of age has little 
effect on the leukemia incidence. 

Dr. R. L. Swarm reports hemangioendothelio- 
mas of the liver and spleen in two of three rabbits 
receiving two injections each of 3 ml. of thorotrast 
(colloidal thorium dioxide). The lesions ap- 
peared at 36-37 months and the other noncancer- 
ous animal died at 19 months. A single intra- 
venous dose of 3 ml. produced no neoplasms in 
any of nine rabbits, four of which survived at 
least 38 months. 

HOST FACTORS IN CARCINOGENESIS 

As better knowledge of constitutional factors 
concerned with the carcinogenic process is ac- 
quired, the interrelationships are more properly 
discussed in association with specific responses 
which they modify under controlled conditions. 
The principal identifiable genetic determinant of 
the cancerous response seems to reside in specific 
organs or tissues as shown in the work already 
reported by Heston, Gardner, Kirschbaum, and 
others. Two strains of mice may be almost com- 
pletely resistant on the one hand, and exquisitely 
susceptible on the other, to production of a given 
anatomical variety of cancer under readily con- 
trollable conditions. In what way do these organs 
differ ? The morphologist, well equipped to study 
this problem, arrives at no definite conclusions. 
The biochemist is confronted with large numbers 
of cells of which an almost insignificant fraction is 
destined to become cancerous. The problem does 
not seem insuperable, however, when one considers 
the availability of microchemical techniques as de- 
veloped by Linderstrom-Lang, Lowry, and others. 

Dr. Heston has observed an increased frequency 
of hepatomas in C3H mice during recent years. 
He ascribes this in part to genetic change in the 
strain, but also to change in the diet in his animal 
colony, since mice eating a standard commercial 
laboratory ration develop significantly fewer tu- 
mors of the liver than those eating a formula es- 
pecially compounded by Dr. Morris for use at 
NCI. The presence or absence of the mammary 
tumor agent in the stock has no influence on the 
incidence of hepatomas. 

The influence of the lethal yellow (Y) gene on 
mammary tumor development studied by Dr. Hes- 
ton is manifested by a prolonged latent period in 



virgin mice of agouti color as compared with those 
having yellow coats. Both color types are ob- 
tained by mating C3H and Y strains since the Y 
gene can be propagated only in heterozygotes. 
The difference in latent period would seem to be 
related to some hormonal influence because it does 
not occur in breeding females. The Y gene also 
affects hepatoma incidence which is greater in yel- 
low mice. An even larger difference is seen when 
the number of hepatomas per mouse is compared. 
Positive correlations exist between frequency of 
tumors of the liver and body weight, body height, 
and length of the femora. 

Dr. M. K. Deringer has previously reported a 
reduction in the incidence of mammary tumors in 
mice of strain C3He which she produced by trans- 
planting fertilized C3H ova to the pregnant uteri 
of C57 black mice. It now appears that DBA/2e 
mice, comparable to C3He, have even fewer breast 
carcinomas. 

Mice of the hairless strain HR develop a con- 
siderable number of skin tumors but principally 
those arising in dermal appendages rather than 
from epidermis. At any rate, they exhibit a 
genetically determined tendency towards tumor 
formation. Ability to enhance chemical carcino- 
genesis in the skin by painting with ethyl carba- 
mate is well-established, though urethane produces 
few if any epidermal tumors by itself. Dr. Derin- 
ger has not been able to influence the production 
of skin tumors in HR mice by topical administra- 
tion of ethyl carbamate. 

Cancers and Their Properties 

Knowledge of neoplastic diseases has profited 
enormously from search by imaginative, diligent, 
and talented scientists for common denominators 
that would permit development of unifying con- 
cepts relating all cancers to one another. At the 
present time everyone would probably agree that 
the transformation from normal to neoplastic is 
basically a heritable and irreversible change in 
cellular behavior — and probably the general ac- 
ceptance of the somatic mutation theory of can- 
cer production means no more than that. Asso- 
ciated with the cancerous change or consequent 
upon it are varying degrees of taxonomic abnor- 
malities in the affected population, and the loss 
to greater or less extent of specialized cellular 
function. The normal tissue of any adult organ 



12 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



has certain definable chemical attributes which 
vary somewhat among individuals of the species. 
Neoplasms arising from these same organs may 
reflect widely different biochemical patterns of ac- 
tivity, and when one compares tumors of different 
histogenesis among themselves the complexity is 
compounded. 

Borst in Germany and Ewing in the United 
States emphasized the diversity of structural pat- 
terns and biological behaviors among cancers aris- 
ing in the same organ. The thesis has been ac- 
cepted by clinicians, and recent experience in 
chemotherapeutic excursions has intensified the 
need for better and different classifications of 
malignant neoplasms than now exist. It seems 
desirable to restudy the biochemistry of cancer to : 

1. Delineate certain qualities measurable by sim- 
ple techniques with a view towards correlating 
rather gross metabolic characteristics with biolog- 
ical behavior, 

2. Establish the degree of change of certain 
specific enzyme activities in neoplasms arising 
from a given organ or tissue, and, 

3. Search for progressive changes in metabolic 
attributes or enzymic patterns as the recognizably 
malignant tumor progresses to an increasingly 
autonomous state. 

A part of this is already in progress. The orien- 
tation of much biochemical activity in cancer is 
concerned with the description of drug action ; yet 
equal concern with the natural history of selected 
cancers as definable in chemical terms might well 
expedite progress in therapeutic research. It is 
unreasonable to expect the biochemist to solve all 
the problems of cancer by himself, and, indeed, a 
better familiarity with neoplastic diseases will re- 
quire development of new techniques and concepts, 
perhaps even new disciplines of science. 

The search for common denominators should 
continue, but there is every reason to focus atten- 
tion on cancers of common origin or different be- 
havior within a histogenetically homogeneous 
group. The final result may reveal entities as dis- 
crete as mumps, chicken pox, and measles within 
one anatomical type of cancer, though one would 
not expect such differences to be associated pri- 
marily with qualitatively different etiologies. 

NEW TUMORS 

The colony of mastomys established by Dr. H. L. 
Stewart is developing a few carcinomas of the 



glandular stomach as expected from experience in 
South Africa. Hepatomas are the most fre- 
quently occurring neoplasms at this time. At- 
tempts to transplant tumors within the species 
have been unsuccessful. 

Dr. Dunn is studying several unusual uterine 
neoplasms with markedly organoid structure 
which arise in strain BALB/C mice. One of these 
after seven months' growth as a transplant pro- 
duces a cystic cavity lined by endometrium with a 
well defined stroma and an outer layer of smooth 
muscle. 

A transplantable osteogenic sarcoma studied by 
Drs. Dunn and Da we contains many large multi- 
nucleated osteoclastic cells which disappear when 
the lesion is grown in vitro but reappear when the 
tissue culture cells are transplanted back to mice. 

Some interesting lesions have been found in Dr. 
Deringer's colony of strain BL mice. The major- 
ity of the females develop large ovaries due to dep- 
osition of amyloid in corpora lutea. The ovaries 
are also one site of a peculiar necrotizing arteritis 
which affects other tissues including the kidneys, 
though its exact distribution and frequency are 
not yet determined. Strain BL, which also de- 
velops some mammary and pulmonary tumors, has 
been reported by Drs. Leon Sokoloff (NIAMD) 
and R. T. Habermann (DRS) to have ideopathic 
necrosis of bone as well. 

The Institute maintains about 175 different 
transplantable tumors, and specimens have been 
sent to all parts of the world. Each represents 
special characteristics, and every new neoplasm 
that can be propagated becomes a useful tool. In 
some cases new tumors generate entirely new pro- 
grams. Research on plasma cell myelomas is a 
case in point. 

PLASMA CELLS AND PLASMACYTOMAS 

Dr. Dunn described a plasma cell tumor arising 
in a C3H mouse during 1954, and maintained it in 
serial transplantation. Dr. Michael Potter in 1956 
commenced an experimental program based on 
availability of this myeloma to which others have 
been added subsequently. His infectious enthusi- 
asm focused the attention of colleagues on the 
value of experimental myelomas as research tools, 
and some information was summarized in earlier 
annual reports. 



NATIONAL CANCER INSTITUTE 



13 



Plasma cells first appear in the mesenteric 
lymphatic tissue of mice at about the 10th day of 
life, according to Dr. K. C. MacCardle. Their 
most distinctive structural feature is a juxtanu- 
clear, clear, cytoplasmic "hof " which contains the 
Golgi apparatus and the centrosome. Plasma 
cells in the 10-day-old mouse have distinctly 
smaller Golgi than those in adult mice. Examina- 
tion of plasma-cell neoplasms leads Dr. MacCardle 
to speculate that the myeloma cells are derived 
from lymphoma-like cells which in turn probably 
originate from undifferentiated lymphocytogenic 
cells in the germinal centers of lymph nodes. 

Dr. Emma Shelton, seeking concrete informa- 
tion on the behavior of normal cells in the presence 
of tumor cells, studied the behavior of normal cells 
when grown in double diffusion chambers placed 
in the peritoneal cavities of mice. Cells cannot 
traverse the walls of these chambers, but fluids 
can. Cells of the peritoneal fluid placed within 
chambers ultimately form an organized tissue con- 
taining typical fibroblasts. Detailed study of the 
sequential events leads Dr. Shelton to conclude 
that lymphocytes grown in this manner modulate 
into an indifferent cell type which has the capacity 
to become either a fibroblast or a plasma cell. 
Macrophages appear to have the same capabilities. 
Granulocytopoiesis occurs in the chambers, and 
most cells persist beyond 140 days. Collagen can 
be identified at 2 weeks and its content then in- 
creases with time. 

Thus two different approaches complement one 
another insofar as the origin of plasma cells is 
concerned, and Dr. Shelton has also awakened a 
long-standing controversy. 

Substantial impetus was given to plasmacytoma 
research through the occurrence of several new 
plasma cell myelomas in mice into which diffusion 
chambers containing mouse mammary carcinoma 
cells had been implanted by the late Dr. G. H. 
Algire and Dr. Ruth Merwin as described in the 
report for 1958. The exact reason for this phe- 
nomenon is still not clear, as some few myelomas 
have now been obtained through implantation 
within the abdominal cavity of empty chambers 
containing no cells, as reported by Dr. Merwin. 
Experiments testing the capacity of various parts 
of the diffusion chambers to initiate the plasma- 
cell tumors have not yet been completed. These 
myelomas have a distinct advantage over the 



earlier plasmacytomas available for study since 
they grow more rapidly. A series of individualis- 
tic plasmacytomas is now available which repro- 
duces most of the features of their human counter- 
part, including circulating myeloma proteins 
characterized by Dr. J. L. Fahey. 

Morphological studies of these neoplasms by 
Dr. MacCardle correlated with biological and bio- 
chemical attributes described by Drs. Potter and 
Fahey reveal three types of Golgi apparatus: a 
few small strands, a diffuse network, or a large, 
thick, and hypertrophied ball-type network. Cells 
of two tumors which produce ^-globulins have 
the third variety, but the type of Golgi seems to 
relate better to the speed of production of myeloma 
proteins than to their electrophoretic properties. 

Dr. A. J. Dalton has examined the ultrastruc- 
ture of 10 transplantable plasma-cell neoplasms. 
Differences within the group relate principally to 
the extent and complexity of the ergastoplasm, 
but these do not correlate well with the physico- 
chemical characteristics of the myeloma globulins. 
A feature common to all of the tumors is the pres- 
ence of minute viruslike intracytoplasmic bodies. 
The particles are formed on the membranes of the 
endoplasmic reticulum, and the membrane itself 
forms the outer shell. Similar bodies are absent 
from normal plasma cells of mice. The signifi- 
cance of the particles is not known, but they may 
well be related to the neoplastic state. They are 
not the mammary tumor agent, as some of the 
myelomas have arisen in agent-free mice and 
bioassay has failed to reveal presence of that virus. 
An attempt to examine cells from human plas- 
macytomas under the electron microscope has been 
beset with a series of technical difficulties which 
are yet to be surmounted. 

Evidence that the myeloma cells secrete the 
myeloma proteins into the serum was published 
in 1958 by Drs. Nathan, Fahey, and Potter. At- 
tention then focused on the relation of these pro- 
teins to those normally occurring in the serum. 
Dr. Fahey has characterized the physicochemical 
properties of human gamma globulins which are 
clearly a large family of related but not identical 
protein molecules. Two major groups with ultra- 
centrifugal sedimentation coefficients of 18S and 
6.6S respectively can be separated by anion-ex- 
change cellulose chromatography. The 6.6S 
group can then be further subdivided into 4 or 



14 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



more fractions. Detailed characterization of these 
fractions reveals a family of molecules sharing 
similar ultracentrifugal and immunochemical 
properties but differing in electric charge, hexose 
content, and antibody activity. 

A similar family of molecules is found when 
myeloma proteins from sera of patients with 
plasmacytomas are examined in detail. These 
myeloma proteins appear to represent proteins 
normally present in serum in small quantities. 
Similar observations have been made on the serum 
myeloma proteins associated with eight trans- 
plantable plasma cell neoplasms. 

Dr. Fahey argues that since the variety of 
myeloma proteins represents a variety of plasma 
cell neoplasms, the spectrum of normal 6.6S 
gamma globulins probably represents a spectrum 
of individual normal but distinctly different 
plasma cells. 

Immune response of myelomatous mice to sheep 
erythrocytes and bovine serum albumin has been 
studied by Dr. Falconer Smith. Capacity to form 
antibodies to either particulate or soluble antigens 
decreases as the tumor grows larger. The sec- 
ondary response remains unaltered and the half- 
life of passively transferred antibodies does not 
change. The results suggest a factory so busy 
forming myeloma proteins that it gives an increas- 
ingly lower priority to antibody production as the 
factory expands. 

Dr. Michael Potter is studying the effect of 
chemotherapeusis on the transplantable myelomas. 
Some of them will regress completely when treated 
with 5-fluorouracil even though initial treatment 
is delayed until the lesions are 2 cm. in diameter; 
others fail to respond. The optimum dose of 
5-fluorouracil is 20 mg./kg. six times weekly. 
Toxicity occurring within 2 to 3 weeks requires 
50 percent reduction in the dose, and the tumors 
recur. Some mice apparently cured of one my- 
eloma have accepted a second graft of the same 
myeloma in contrast to Dr. Abraham Goldin's 
experience with mice cured of leukema L1210 by 
treatment with 3', 5'-dichloroamethopterin (vide 
infra). 

New plasmacytomas will doubtless appear in 
the course of time and be added to the fine col- 
lection now available. The progress made to date 
already permits an aggressive attack on specific 
problems concerned with causation and treatment, 



and also emphasizes the usefulness of these tools 
in pursuing basic problems in protein chemistry 
and metabolism. All logical reasoning requires 
a considerable expansion of the chemotherapeutic 
effort. The individual tumors faithfully repro- 
duce the manifestations of multiple myeloma as 
seen in the clinic, including some variety of in- 
dividualistic behavior patterns. 

While plasmacytoma is rather well-defined in 
biological and chemical terms, macroglobulinemia 
occurs also under more poorly understood circum- 
stances. Dr. Fahey has studied gamma macro- 
globulinemia in patients with a variety of dis- 
eases, including the syndrome described by Pro- 
fessor Jan Waldenstrom of Malmo, Sweden, who 
visited the Institute during February, and the 
Bing-Neel syndrome. Correlative studies by Dr. 
T. F. Dutcher of lesions among those patients with 
cancer and macroglobulinemia have revealed char- 
acteristic lymphocytoid plasma cells with intra- 
nuclear inclusions. Drs. Dutcher and Fahey sug- 
gest that the intranuclear periodic-acid-Schiff 
positive material and intranuclear vacuoles seen 
on phase contrast microscopy are chemically iden- 
tical to the circulating 18S, hexose-rich, y-macro- 
globulin. Sequential cellular changes among the 
lymphocytoid plasma cells suggest those cells as 
the site of macrogobulin formation. The investi- 
gators regard macroglobulinemia as a neoplasm of 
reticuloendothelial origin, a variant of multiple 
myeloma, or a type of lymphoma. 

Blood viscosity may be markedly elevated in 
macroglobulinemia and was the immediate cause 
of cardiac failure in one of Dr. Fahey's patients. 
He has designed a therapeutic regimen for main- 
taining the blood viscosity below the critical level. 

DISSEMINATION OF CANCER 

The ability of cancerous cells to invade sur- 
rounding tissues and colonize distant parts of 
the body through access to vascular channels is 
the most important factor limiting the effective- 
ness of surgical or radiological therapeusis. 
While the routes of dissemination are reasonably 
well understood, the basic biological facts responsi- 
ble for metastasis are obscure, especially since 
metastasizability may be an attribute acquired 
after the transformation to the cancerous state 
has occurred. 



NATIONAL CANCER INSTITUTE 



15 



Dr. Shelton is studying experimental lymphoid 
neoplasms which, on transplantation, remain lo- 
calized or disseminate rapidly. Difference in the 
capacities of these neoplasms to penetrate the walls 
of diffusion chambers has been described in earlier 
reports. Normal cells of the peritoneum and in- 
vasive lymphomas L1210 or L2 grow independent- 
ly of one another when placed in diffusion cham- 
bers. The growth of such non-invasive lymphoma 
cells as LI and P353, however, tends to be asso- 
ciated with growth of the peritoneal cells, since 
the tumor cells grow in definite clumps, sur- 
rounded and enclosed by normal cells. Fibro- 
blasts from subcutaneous fat pads of the mouse 
grow luxuriantly in diffusion chambers, and Dr. 
Shelton has introduced neoplastic cells into the 
fibroplast population. Again, cells from L1210 
grow as free-floating independent entities. 
Growth of LI cells is intimately related to pro- 
liferating fibroblasts, and these tumor cells never 
grow beyond the advancing edge of the fibroblast 
colony. 

Study of circulating tumor cells in cancer pa- 
tients proceeds as a joint effort of the Surgery 
Branch, the Laboratory of Pathology, and the 
PHS Hospital in Baltimore, led by Drs. K. R. 
Smith, R. A. Malmgren, and J. F. Potter. Can- 
cer cells are found more often in the efferent blood 
from a malignant neoplasm than in blood taken 
from the antecubital vein. This was to have been 
expected and could relate almost solely to dilution 
effects, though the filtering action of the liver, 
lungs, and other organs cannot be excluded at this 
time. Patients whose cancers are apparently 
amenable to definitive treatment have tumor cells 
in peripheral blood about half as often as do those 
whose disease is recognizably disseminated. 

Circulating tumor cells have been found in pe- 
ripheral venous blood in about 75 percent of 
patients with malignant melanoma. The cells are 
seen sporadically in serial specimens even though 
the melanoma is widespread. Only two of six 
patients whose melanomas were surgically resect- 
able had recognizable cells in the peripheral blood 
preoperatively. Both of them rapidly developed 
recurrent disease and died. 

The significance of circulating tumor cells will 
become apparent only after a long comprehensive 
study of specific anatomical types of cancer in in- 
dividual patients. Correlated animal studies are 



in progress, but the same methods by which one 
prepares specimens of human blood cannot be ap- 
plied to mouse blood for some obscure reason, and, 
of course, the small size of most laboratory ani- 
mals that develop cancer in a reasonably predict- 
able fashion or will support the growth of trans- 
planted neoplasms militates against the study of 
serial specimens from the same individual. 

Other studies of metastasis in experimental ani- 
mals have been especially rewarding. Housing 
tumor-bearing mice individually has produced a 
marked reduction in the variability of results as 
far as metastases studies are concerned. The abil- 
ity to identify and count minute deposits of non- 
pigmented tumor cells in the lungs has been greatly 
improved by Miss Hilda Wexler, who introduces 
a solution of india ink into the trachea, thereby 
increasing the contrast between normal and can- 
cerous tissue. 

A much larger experience in the study of pul- 
monary metastases produced from transplanted 
tumors growing in the thighs of genetically ap- 
propriate mice points to a uniform type of be- 
havior among the neoplasms investigated. Dr. 
A. S. Ketcham reports definite reduction in the 
number of metastases when the tumor-bearing ex- 
tremity is amputated as compared with control 
mice whose tumors are either undisturbed or par- 
tially excised. The mean size of the pulmonary 
metastases in unit time is greater among the mice 
with amputated legs than among controls. The 
previous divergent results obtained by Dr. W. E. 
Schatten seem to relate to experimental design. 

Transplantable mouse melanoma S-91 has been 
a most useful tool in these studies. Several dif- 
ferent specimens of the tumor have been obtained 
from Dr. M. W. Woods who propagates the neo- 
plasm by trocar transplantation, and pulmonary 
metastases seem to have been rather infrequent. 
Dr. Ketcham and associates find it necessary to 
transplant cytosieved preparations of S-91 mela- 
noma through 7 to 9 serial passages before a re- 
producible pattern of metastasis develops which 
can be quantified. This tantalizing experience 
may be extremely important since it suggests that 
some factor of selection may be involved in the 
production of metastasizing melanomas and that 
only some of the cellular population of this par- 
ticular neoplasm are capable of metastasizing. 
This phase of the program will be extended. 



16 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Equally fascinating is an observation on factors 
involved in the localization of metastases as re- 
ported by Dr. D. L. Kinsey. Last year's report 
referred to the predilection of the lungs for cells 
of melanoma S-91 introduced into either arteries 
or veins despite the ability of the same cells to 
grow when introduced directly into various or- 
gans and tissues. (Metastases can be produced in 
the liver when large, approximately lethal, doses 
of S-91 cells are introduced into tributaries of the 
portal vein.) Dr. Kinsey transplanted pieces of 
lungs to the thighs of genetically suitable mice and 
observed growth of melanoma cells in both the ec- 
topic and normal pulmonary tissue when the tu- 
mor cells were injected into the blood stream. 
Transplants of other organs and tissues to the 
thigh would not support growth of intravascu- 
larly injected cells of melanoma S-91. This seems 
to be the first experimental confirmation of the 
thesis ascribing certain bizarre patterns of meta- 
stasis to a locus minoris resistentiae. In this par- 
ticular example the basic mechanism may well re- 
late to the known capacity of the lungs to filter 
cells from the circulating blood. 

Dr. A. H. Harris' attempts to standardize the 
growth of a transplantable hamster melanoma 
provided by Dr. J. H. Edgcomb have not been 
successfvd. 

Experiments concerned with chemotherapeutic 
prevention of metastasis formation or treatment 
of artificially produced pulmonary metastases 
have produced no significant new findings. On 
the other hand, Dr. R. C. Hoye finds that treat- 
ment of three out of four different experimental 
mouse tumors with 7l5r X-irradiation reduces the 
incidence of pulmonary metastases by 70 percent 
and a dose of 2,000r effects 81-100 percent reduc- 
tion. 

POLYSACCHARIDE RESEARCH 

A long-term interest in polysaccharides at the 
National Cancer Institute has stemmed from the 
observation by Dr. M. J. Shear and his colleagues 
that this class of macromolecule can damage both 
clinical and experimental neoplasms. Certain 
problems arising repeatedly over the years re- 
quired some expansion of this research area dur- 
ing 1956, designed especially to broaden its 
scope, especially at a fundamental level. Substan- 



tial information has been obtained regarding 
many important aspects of lipopolysaccharides. 

Bacterial endotoxins are important members of 
this chemical species. During the year, Dr. Mau- 
rice Landy and Dr. Edgar Bibi (NT AID) have re- 
duced the lipid content of lipopolysaccharides 
from 19.2 to 1.2 percent without reduction in their 
toxicity and without detectable effect on endotoxic 
properties. Dr. Woods reports that bacterial 
endotoxins in concentrations of 0.003 to 0.3 p.p.m. 
stimulate aerobic glycolysis of cells from mela- 
noma S-91, and correlates the degree of stimu- 
lation with the degree of activity in vivo, as deter- 
mined by Dr. Landy. 

The endotoxin detoxifying component (EDC) 
discovered by Dr. Landy, which inactivates poly- 
saccharides with respect to tumor necrosis and 
stimulation of specific antibody formation, also 
acts in the same way against killed S. typhosa but 
not against the viable organisms. The latter can 
be inactivated by sera from several species by a 
different bactericidal system. Normal antibody, 
all four recognized components of complement, 
and divalent cations are required for the bacteri- 
cidal systems. The concomitant elimination of 
endotoxic attributes of typhoid bacilli in the bac- 
tericidal process of normal serum has obvious 
important implications for the host-parasite rela- 
tionship. 

Plasma of febrile rabbits that have been treated 
with bacterial lipopolysaccharides has been re- 
ported to contain "endogenous pyrogen," a sub- 
stance with fever-producing properties quite dif- 
ferent from those of the polysaccharide injected 
originally. Since reduction in the pyrogenicity of 
endotoxin following incubation in vitro with nor- 
mal human serum was reported years ago, Dr. 
Landy undertook a study to compare the prop- 
erties of "endogenous pyrogen" with endotoxins 
incubated with serum. The very properties used 
to distinguish "endogenous pyrogen" from poly- 
saccharide are those which appear after incuba- 
tion of the macromolecules in serum with respect 
to: 

1. Fever production in endotoxin-tolerant 
animals. 

2. Dissociation of leukopenia from the febrile 
response. 

3. Loss of tolerance to daily injections of altered 
endotoxins. 



NATIONAL CANCER INSTITUTE 



17 



4. Failure to develop acute refractoriness to 
multiple injections given over a short period of 
time. 

Strains of E. coll vary in their susceptibility 
to the bactericidal system in serum. Study of 
two widely divergent strains by Dr. Landy and 
Dr. J. G. Michael (Visiting Scientist) reveals that 
the concentration of polysaccharide is inversely 
related to susceptibility of the organism to the 
bactericidal system. Cells and extracts of the 
resistant strain exhibit greater endotoxic potency 
as measured by ability to necrose sarcoma S37. 
Normal human and rabbit sera contain low con- 
centrations of natural antibodies specific for 
bacteria of several genera of the Enterobacteria- 
ceae. Their bactericidal action is not increased 
for E. coli by addition of a specific immune anti- 
body. 

These observations have led Dr. Landy to search 
for substances in normal serum which affect the 
viability of tumor cells. Incubation of cells 
from sarcoma S37 with normal human serum 
abolishes their capacity to grow in mice. Meta- 
bolic activity, as measured in Warburg flasks by 
Dr. Woods, is lost. The cells no longer reduce 
neotetrazolium. They stain readily with eosin, 
and blebbing of the cell membranes occurs. In 
short, they are dead. The effect of the tumoricidal 
action can be eliminated by incubation of 52° C. 
for 30 minutes or by adsorption with washed sar- 
coma cells at 4° C. Original activity is restored by 
combining the heat-inactivated serum with the ad- 
sorbed one, indicating that two factors are con- 
cerned with the tumoricidal activity. All four 
components of complement and divalent cations 
are needed. Tumoricidal effect is undiminished 
at dilutions of 1 : 8 but is decreased at 1 : 16. One 
milliliter of serum will destroy 30 million cells in 
two minutes at 37° C. Although this factor is 
widespread in nature, it is not detectable in the 
sera of all species, nor has it any noticeable effect 
when studied in vivo. Cells of several different 
mouse tumors are killed by the tumoricidal factor, 
but the only normal cells which exhibit similar 
sensitivity are reticuloendothelial. 

A cytolytic factor in human serum has been de- 
scribed by Dr. B. Bjorklund (State Bacteriologi- 
cal Laboratory, Stockholm, Sweden) . The agent 
is normally coupled with an inhibitor from which 
it can be dissociated by dialysis and obtained in a 



reasonably pure state by electrophoresis. The iso- 
lated factor lyses four established strains of hu- 
man cells in vitro. Dr. Bjorklund visited the Na- 
tional Cancer Institute late in the year to compare 
his agent, which also produces marked changes in 
a variety of experimental ascites tumors, with the 
agent described by Dr. Landy. They appear to 
be quite different. 

The chemical component of the polysaccharide 
research program is led by Dr. P. T. Mora. Poly- 
glucoses and their derivatives are being used to 
study fundamental aspects of enzyme-substrate in- 
teractions (vide infra) and are in great demand by 
extramural scientists. The majority of the deriva- 
tives are negatively charged, but during the year 
Dr. Mora and Mr. J. W. Wood have prepared posi- 
tively charged synthetic polyglucose derivatives to 
study the inhibition of biologically active anionic 
macromolecules. The synthetic method of prepar- 
ing polysaccharides by chemical polycondensation 
procedures is being extended to numerous other 
sugars. High molecular weight polymers of 2-de- 
oxygiucose, ribose, rhamnose, galactose, maltose, 
mannose, arabinose, etc., have been obtained in 
good yield. 

A new, large-scale polysaccharide preparation 
designated P-45, made by Mr. Adrian Perrault 
from S. marcescens, resembles the earlier P-25 ma- 
terial obtained from the same organisms. Dr. 
Ezio Merler (Visiting Scientist) finds P-45 elec- 
trophoretically homogeneous, although two com- 
ponents with different sedimentation coefficients 
appear in the ultracentrifuge. The heavier com- 
ponent seems to be an aggregate of the lighter one. 
P^5 is a strongly negative macromolecule. Titra- 
tion of negative groups, in collaboration with Dr. 
H. A. Saroff (NIAMD), reveals a pKa of about 
2.7, most likely due to carboxyl groups of the 
neuraminic acid type. The great bulk of the poly- 
saccharide is a polymer of glucose containing 0.3 
percent phosphorus in some organically bound 
form and an extremely small amount of amino 
acids. 

The ability to bind certain molecules to syn- 
thetic substituted polyglucoses by electrostatic 
forces and to release the bound molecule by adding 
a second macromolecule with a stronger charge 
seems to apply to many different enzymes. For 
example, the most important factor in inhibiting 
ribonuclease activity with the macromolecules of 



18 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



interest is the charge density as measured by de- 
gree of sulfation. The maximum sulfation is 3 per 
anhydrogiucose unit which produces more than 
1,000 times the inhibition possible with heparin, 
the best agent previously described possessing sim- 
ilar activity. Charge density seems to be the most 
important factor in enzyme inhibition also- with 
carboxyl derivatives of polyglucose. The molecu- 
lar weight of the inhibiting substance must be at 
least 2,000, but the internal configuration of the 
molecule appears to be less important. 

Other physicochemical studies carried out by 
Dr. Mora have provided further information on 
enzyme-substrate interaction involving two oppo- 
sitely charged macromolecules. Titration of poly- 
cations with polyanions follows stoichiometric 
equivalence. Such interactions differ from those 
involving low molecular weight ions, since a firm 
electrostatic complex forms under certain condi- 
tions, leading to precipitation of the macromolec- 
ular components. The precipitation phenomenon 
can be used to fractionate macromolecular sub- 
stances and the principle has been used in concen- 
trating EDC activity from human serum by Dr. 
Mora and Mr. B. G. Young. 

Administration of polymyxin, neomycin, strep- 
tomycin, or protamine to mice in doses which cause 
death within 25 to 40 minutes can be counteracted 
by a neutralizing equivalent of anionic synthetic 
polysaccharide derivative administered by the 
same intraperitoneal route if given 10 minutes 
after the toxic dose of the cationic antibiotics as 
reported by Dr. Mora, Mr. Young, and Dr. Shear. 
Prior subcutaneous injection of the antidote pre- 
vents death from large intraperitoneal doses of 
cationic antibiotics. Again, the most highly 
charged polyglucose derivatives are the most effec- 
tive antidotes in this system. The same type of 
approach has been used to inhibit the activity of 
T2 phage. 

These studies of macromolecular interactions 
which block important biological activities are 
extremely important not only in relation to the 
nature and mechanism of nonspecific resistance as 
well as immunologic factors concerned with can- 
cer, but also in providing a new method for sep- 
arating biologically active compounds. 

Inactivation of a polysaccharide by electron 
bombardment has been studied by Drs. J. W. 
Preiss and Morris Belkin. Progressive inactiva- 



tion occurs with increasing dose as measured by 
decreasing ability of the molecule to produce 
vacuolation in tumor cells growing in vitro. Inac- 
tivation of this polysaccharide takes 16 times the 
dose required to attenuate the sucrose-hydrolyz- 
ing activity of invertase. Since the densities of 
these molecules are similar, the molecular weight 
of the active site of the polysaccharide is of the 
order of 7,500. 

PROTEIN AND AMINO-ACID CHEMISTRY 

The chromatographic separation of soluble pro- 
teins as initiated by Drs. H. A. Sober and E. A. 
Peterson has had a profound effect on the develop- 
ment of biochemistry throughout the world. Iso- 
lation of antibodies from serum is readily accom- 
plished by a simple, rapid procedure, described 
by Dr. Sober, Dr. Peterson, and Dr. H. B. Levy 
(NIAID), which recovers about 90 percent of the 
gamma globulins free of macroglobulins. The 
macroglobulins can be obtained by zone electro- 
phoresis of the remaining proteins. 

Chromatography is also being used in collabora- 
tion with Dr. B. L. Vallee (Peter Bent Brigham 
Hospital, Boston) , to isolate metal-containing pro- 
teins. The same metal may occur in distinctly 
different fractions. 

Drs. Peterson and Sober can produce extensively 
resolved and reproducible chromatograms of se- 
rum or plasma in 6 hours. Limitation of flow 
rate is hydrodynamic, but resolution is not greatly 
affected by the highest flow rates permitted by the 
mechanical properties of the adsorbents used. 
Buffer exchange on Sephadex provides some ad- 
vantages over dialysis as a means of equilibrating 
protein and buffer required at the start of a chro- 
matographic experiment. Buffer exchange per- 
mits salt displacement chromatography which in- 
creases adsorptive capacity of the column and 
permits new approaches to stepwise displacement 
to overcome objections inherent in ordinary step- 
wise elution. 

The principal problem created by protein chro- 
matography is the considerable variety of physi- 
cochemically distinct entities with the same or 
similar biological or immunological properties 
(see Annual Report 1958). Drs. Peterson and 
Sober report an increase in the heterogeneity of 
serum proteins when treated with 8 M urea as 



NATIONAL CANCER INSTITUTE 



19 



measured by electrophoretic analysis of the chro- 
matographically separated peaks. The urea treat- 
ment, however, does not alter the chromatographic 
elution profile significantly. Further study of 
complexing of human serum mercaptalbumin with 
lipids reveals a markedly increased affinity of the 
protein for DEAE-cellulose. Addition of a cati- 
onic lipid, however, can abolish affinity of the pro- 
tein complex for this adsorbent but increases its 
adsorption to CM-cellulose, indicating that the 
lipid-protein complex is highly basic. 

Treatment of alkaline solutions of an amino acid 
with an aldehyde in the presence of copper ions 
as described by Drs. T. T. Otani, Nobuo Izumiya 
(Visiting Scientist) , S. M. Birnbaum, and Milton 
Winitz proceeds with the formation of the corre- 
sponding a-amino-/?-hydroxy acid. The funda- 
mental reaction has permitted single-step prepa- 
ration of : 

1. /Mrydroxy-/3-methylaspartic acid from py- 

ruvic acid and glycine. 

2. /3-hydroxyaspartic acid from glyoxylic acid 

and glycine. 

3. /3 -hydroxy leucine from isobutyraldehyde and 

glycine. 

4. Serine from formaldehyde and glycine. 
Evidently the method is a general one providing 
a simple and economical route to the production 
of a-amino-y8-hydroxy amino acids useful in bio- 
chemical research. 

Dr. Marco Rabinovitz, interested in the inhibi- 
tion of protein synthesis by amino acid analogs, 
has studied several in which the methylene group 
in the side chain has been replaced by sulfur. 
Methionine sulf oxamine reduces by 50 percent the 
rate of protein synthesis in washed Ehrlich as- 
cites cells through prevention of glutamine syn- 
thesis. The inhibition of protein synthesis by the 
mixed disulfide of methylmercaptan and cysteine 
in low concentrations is prevented by glutathione, 
but not by methionine. Dr. Rabinovitz reports 
interference of incorporation in vivo of isotopi- 
cally labeled lysine into the tumors and livers but 
not the spleens of cancerous mice by administra- 
tion of the lysine antagonist 4-thialysine. 

Drs. K. Michi (Visiting Scientist), Birnbaum, 
and Winitz have separated diastereomeric mix- 
tures of amino acids such as isoleucine-alloisoleu- 
cine, hydroxypropoline-allohydroxyproline, and 

556044—60—3 



threonine-allothreonine by chromatography on 
Amberlite columns in amounts as great as 20 
grams. Recovery is 90-95 percent. The program 
initiated by the late Dr. Greenstein for the pro- 
duction of optically active, pure amino acids in 
adequate amount and at reasonable cost to pro- 
vide powerful tools for research in the chemistry 
and metabolism of amino acids and proteins has 
been completely successful. 

Drs. Winitz and Birnbaum, with Mr. M. C. 
Otey and Drs. T. Sugimura and V. Mitbander 
(Visiting Scientists), have commenced to use 
chemically defined diets to study specific enzymes 
and metabolic processes in the intact rat. Earlier 
workers having shown that the Z>-isomers of the 
essential amino acids methionine, tryptophan, and 
leucine can replace, in whole or in part, Z-isomers 
of the same compounds in rats, the subject was re- 
studied with the chemically defined rations. 
Methionine was the only one of the group in 
which weight increment in weanling rats was 
equally good on D and Z isomers. Z> -tryptophan 
was only partially effective in replacing its Z an- 
tipode, and 1.4 times the amount of Z-leucine was 
required before any growth occurred. A series of 
diets was designed to discriminate between the 
relative capacities of Z-amino acid oxidase to con- 
vert the D -amino acids to corresponding a-keto 
acids and of transaminases to accomplish the 
transformation of a-keto acids to corresponding 
Z-amino acids. All possible combinations of the 
isomers of alanine and methionine were em- 
ployed, but only the diet using D forms of both 
amino acids produced marked retardation in 
weight increment. 

Other investigators had shown sodium benzoate 
to be a potent inhibitor of Z-amino acid oxidase 
activity in vitro. Synthetic diets containing 2 
percent sodium benzoate and Z ) -methionine pro- 
duced strikingly diminished rates of weight gain, 
according to Dr. Winitz and associates. These 
data point to oxidative deamination as rate limit- 
ing, and studies on the effects of the a-keto analog 
of methionine are in progress. One additional 
point of interest is the sparing effect of dietary 
glycine on the inhibition of Z-amino acid oxidase 
by sodium benzoate through its removal as hip- 
puric acid. 

A long study designed to produce a chemically 
denned diet suitable for human use has been com- 



20 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



pleted as a joint effort of the Laboratory of Bio- 
chemistry and the Surgery and General Medicine 
Branches, with Dr. R. B. Couch serving as the co- 
ordinator. The earlier diets were semisynthetic 
and contained racemic mixtures of amino acids as 
the only source of nitrogen, and the general com- 
position conformed to recommendations published 
by the Food and Nutrition Board of the National 
Research Council insofar as they were available. 
Substantial progress was made, however, from the 
experience obtained by nutritional studies in rats 
by Drs. Greenstein, Winitz, Birnbaum, and Mr. 
Otey. The source of carbohydrate gave consider- 
able difficulty because excessive sweetness pro- 
duced nausea. Dextrose finally proved to be the 
best carbohydrate and, in fact, it was found ad- 
visable to add a flavoring material if a high 
dextrose diet is to be taken by mouth. The prob- 
lem of frequent small watery stools with any of 
the soluble diets was solved by addition of car- 
boxymethylcellulose. The best diet, which con- 
tains L rather than J9-amino acids, can be stored 
in dry form and dissolved as needed; fat-soluble 
vitamins and ethyl lineolate mixed with polysor- 
bate 80 to form a reasonably stable emulsion are 
added and mixed. 

Seven patients have received 12 separate trials 
on the chemically defined diets, five by indwelling 
esophagostomy tubes, and two orally. After it 
had become possible to feed a diet for 7 to 12 days 
without weight loss in an asymptomatic individual 
whose weight had been stable for some time, the 
critically important experiments were done by 
Drs. D. M. Watkin and L. E. Rosenberg on two 
patients under complete metabolic control. These 
studies revealed a need for approximately 25 per- 
cent increase in caloric intake over that provided 
by an isonitrogenous diet of natural foodstuffs in 
order to maintain weight and permit nitrogen 
retention after the period of nitrogen loss induced 
by a nitrogen-free diet. The experience in 
patients coincides with the findings of Dr. Birn- 
baum that the best chemically defined diet is in- 
ferior to diets made of whole foods and supports 
the contention of Rose that diets containing only 
pure amino acids as the source of nitrogen require 
a greater caloric intake for efficiency comparable 
to isonitrogenous diets of whole foods. 



NUCLEIC ACID RESEARCH 

Complex interrelationships among the purines, 
pyrimidines, and chemical intermediates involved 
in the production of polynucleotides are slowly 
being defined, and the contributions by scientists 
of the National Cancer Institute are steadily in- 
creasing. Drs. W. C. Schneider and Jean Roth- 
erham, continuing their studies of deoxyribosidic 
compounds, have defined factors concerned with 
urinary excretion of these substances. Deoxycyti- 
dine is the major deoxyribosidic compound found 
in the urine of rats ; deoxyuridine and an uniden- 
tified compound are also present. These materials 
are not derived from dietary sources, since neither 
fasting nor the use of chemically defined diets, 
through the cooperation of Dr. Winitz, has much 
influence on the level of excretion. The germ- free 
state also has no pronounced effect; hence, bac- 
terial synthesis in the gastrointestinal tract cannot 
be a major factor in their production. Excretion 
of urinary deoxyribosidic compounds usually de- 
clines for 2 or 3 days following partial hepa- 
tectomy and also decreases during the most rapid 
growth phase of the Novikoff hepatoma. These 
two responses, however, are not entirely constant. 

Incubation of hepatoma cells with isotopically 
labeled deoxycytidine results in progressive label- 
ing of deoxyribonucleic acid. Dr. Schneider also 
finds an amount of radioactivity in the nucleotide 
fraction sufficient to suggest the presence of 14 or 
more deoxynucleotides of which some contain 
deoxycytidine. Administration of labeled de- 
oxycytidine to the rat results in uniform distribu- 
tion of C 14 throughout the body ; the amount of ra- 
dioactivity in any given tissue is so low as to dis- 
courage attempts at isolation of the labeled com- 
pounds. Dr. J. H. Weisburger has collaborated 
with Dr. Schneider in the synthesis of phospho- 
ethanolamine-l,2-C 14 , and synthesis of the phos- 
phocholine analog is in progress. Deoxycytidine, 
containing analogs of these compounds, was iso- 
lated from the Novikoff hepatoma, and a study of 
their function seems desirable. 

Dr. R K. Kielley has encountered difficult tech- 
nical problems in her studies of enzymes involved 
in synthesis of deoxyribonucleic acids by normal 
and neoplastic cells. Older techniques for isola- 
tion of nuclei have required substantial modifica- 



NATIONAL CANCER INSTITUTE 



21 



tion for use with cex-tain hepatoma cells, and sepa- 
ration of diesterase and nucleotidase activities in 
snake venom has been accomplished. 

Keports from several sources of major quantita- 
tive changes in urinary excretion of purines and 
pyrimidines during chemotherapeutic treatment 
of acute leukemia encouraged a collaborative pro- 
gram, led by Dr. J. C. Reid of the Laboratory 
of Physiology with colleagues in the General 
Medicine Branch, to isolate and identify purines, 
pyrimidines, and intermediates presumably asso- 
ciated with nucleic acid synthesis and/or degra- 
dation. Chromatographic procedures have been 
used extensively by Dr. Reid in devising analyti- 
cal methods for detection and quantification of 
nucleotides, nucleosides, purines, and pyrimidines 
in human urine. Most difficulties have been obvi- 
ated during the past two years, but xanthosine 
recovery is still low and the ammonium acetate 
gradient used for elution of the major nucleoside 
fraction does not yet give good reproducibility. 
Quantification of all individual components at 
the present time is an arduous and complicated 
task that would seriously limit the usefulness of 
the method for study of disease. Dr. A. W. Pratt 
is working with Dr. Reid to automate the process 
to the fullest possible extent. One approach now 
being pursued is a computational analysis of mul- 
ticomponent spectra applying several mathemati- 
cal techniques made practical by the digital com- 
puter. 

Methods for isolation of nucleic acids and the 
determination of their chemical and physical 
properties are improving. These large molecules 
are sometimes rather fragile, as are proteins, and 
comparatively small insults during isolation may 
denature the molecule. Dr. Joseph Shack finds 
the binding of deoxyribonucleic acids by mag- 
nesium ions essentially independent of pH in the 
range where no titration of nucleate occurs. The 
quantity of magnesium bound is proportional to 
the concentration of deoxyribonucleic acid at a 
given concentration of magnesium ions. 

Evidence presented last year by Dr. Shack and 
Dr. Kilham (DBS) suggested that the transform- 
ing agent prepared from rabbit myxoma virus con- 
tained deoxyribonucleic acid. Attempts to isolate 
an active deproteinized nucleic acid by the phenol 
procedures have been unsuccessful. The trans- 
forming factor prepared from myxoma virus by 



heat alone is active in such various tissue cultures 
as squirrel and monkey kidney, rat embryo, and 
cells from the kidneys of either cottontail or do- 
mestic rabbits. It can enter the cell in the absence 
of fibroma virus where its activity can be detected 
for as long as 3 days. Transforming factor pre- 
pared by a procedure using both heat and urea is 
active only in tissue cultures of rabbit kidney, the 
natural host of both fibroma and myxoma viruses, 
but this preparation is destroyed in the cell within 
three hours of its entry. 

Fractionation of the Novikoff hepatoma by Dr. 
E. L. Kuff using gentler physical procedures in 
conjunction with electron microscope studies by 
Dr. R. F. Zeigel has permitted isolation of free 
nucleoprotein particles representing 50 percent of 
the total ribonucleoprotein in that neoplasm. The 
spherical particles occur in several size groups, 
the most abundant of which measure 24 m//, in 
diameter and have a molecular weight of 4.5 mil- 
lion. Addition of adenosinetriphosphate in con- 
centrations comparable to those found in tissues 
causes dissocation of the particles into smaller 
subunits. Adenosinediphosphate is less effective 
and treatment with deoxycholate, the detergent 
usually used for isolating ribonucleoprotein par- 
ticles, removes one-third of the protein without 
changing the spherical form. Dr. Kuff suggests 
that the physical form of ribonucleoprotein may 
relate to the metabolic state of the cell. 

Dr. M. E. Maver has previously described the 
hydrolysis of cyclic adenylic acid to 2' adenylic 
acid by a ribonuclease preparation obtained from 
spleen. This type of activity has been separated 
by chromatography from acid ribonuclease activ- 
ity. The ribonuclease activities of rat lymphosar- 
coma closely resemble those of calf spleen. The 
acid ribonucleases of rat liver and hepatoma hy- 
drolyze only cyclic pyrimidine nucleotides to give 
the 3' nucleotides. The corresponding acid ribo- 
nucleases obtained from spleen or lymphosarcoma 
hydrolyze both purine and pyrimidine cyclic nu- 
cleotides to yield 2' derivatives before purification 
by chromatography. 

Dr. Maver also describes some differences in de- 
oxyribonucleases isolated by the same procedure 
from different tissues. Those obtained from calf 
spleen and liver have a sharp pH optimum at 4.8, 
whereas the deoxy ribonuclease from rat lympho- 
sarcoma acts equally well from pH 4 to pH 5.8. 



22 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Trypsin destroys a substance obtained from hu- 
man leucocytes which inhibits the activity of de- 
oxyribonuclease I but does not affect the enzyme. 
Destructive action of trypsin is prevented by soy- 
bean trypsin inhibitor which does not prevent de- 
struction of the inhibitor by a factor present in 
normal human serum. These findings have pro- 
vided Dr. Shack a basis for assaying the activity 
of both deoxyribonuclease I and the inhibitor and 
to show that normal human urine contains large 
quantities of the enzyme but no soluble inhibitor. 

Dr. E. P. Anderson, working with Dr. L. A. 
Heppel (NIAMD), has studied a new phospho- 
diesterase, which hydrolyzes certain 5 '-phosphate- 
ended ribopolynucleotides to mononucleoside-5'- 
phosphates. Individual nucleotides are released 
serially from the end of the polymer. The enzyme 
is active against ribo-oligonucleotides but not 
against simple esters of nucleoside-5'-phosphates, 
because of a need for a nucleoside moiety on each 
side of the susceptible phosphodiester bond. En- 
zymes with similar hydrolytic activity are found 
in tumors. 

Drs. Sober and Peterson and Dr. Matthias 
Staehelin (Visiting Scientist) report that oligonu- 
cleotides prepared by digestion of ribonucleic 
acids with pancreatic ribonuclease can be frac- 
tionated on cellulose anion adsorbents using vola- 
tile salts for elution. Fractionation is based 
primarily on differences in size of the molecules, 
but the order of emergence from the column also 
relates to basic composition and sequence. Where- 
as other methods permit separation of tetra- or 
pentanucleotides, column chromatography permits 
fractionation of decanucleotides and even larger 
molecules, encouraging further study of specificity 
of interactions of nucleic acids and enzymes. 

ENDOCRINOLOGY 

Extensive metabolic studies conducted by Drs. 
D. M. Bergenstal and M. B. Lipsett with hypo- 
physeal growth hormone from beef, whale, and 
sheep treated with chymotrypsin have revealed no 
characteristic activity in man. Purified and un- 
treated growth hormone from these species is 
similarly inactive. Human growth hormone it- 
self is a potent anabolic agent and increases the 
daily excretion of urinary calcium from 20 mg. to 
150 mg. daily. 



Administration of growth hormone to three 
patients with acromegaly, reported by Drs. 
Bergenstal and Lipsett, caused no retention of 
nitrogen, though the excretion of calcium in the 
urine increased. Testosterone did produce an 
anabolic effect in the same patients; hence, dif- 
ferent mechanisms must be concerned with nitro- 
gen retention on administration of growth hor- 
mone and testosterone, respectively. 

Drs. Bergenstal and Lipsett describe significant 
catabolic effects of small doses of Cortisol and tri- 
iodothyronine in patients with panhypopituitar- 
ism. They emphasize the necessity for defining 
physiological effects of hormones in relation to 
endocrine status of the patients treated. 

A study of the initial stages of stimulation of 
the adrenal cortex with ACTH has been com- 
pleted by Dr. Lipsett and Dr. H. N. Wilson 
(NIAMD). Biosynthesis of adrenal androgens 
depends less on ACTH than does the biosynthesis 
of Cortisol. Study of the precursors of dehydro- 
epiandrosterone by these scientists has encountered 
difficulty because of problems involved in identify- 
ing a metabolite. 

Dr. Lipsett has completed a study of hirsutism 
in women without establishing any correlation of 
extent or severity of hirsutism with the excretion 
of Ci 9 2 or C19O3 metabolites or of pregnanetriol. 

The availability of progestational compounds 
free of estrogenic activity which act on oral ad- 
ministration has permitted examination of the 
effects of large doses of progestin by Drs. Bergen- 
stal and Lipsett. Pro vera (6-methyl 17-acetoxy- 
progesterone), in contrast to progesterone, has 
caused negative nitrogen balance only once in 
seven trials. Provera has no antialdosterone 
activity, nor does it suppress gonadotropin excre- 
tion in postmenopausal patients or in girls with 
precocious puberty. Administration of this 
progestogen causes large increases in the excretion 
of Porter-Silber chromogens; hence, the urinary 
metabolites were partially characterized. Known 
functional groups concerned with the Porter- 
Silber reaction have been ruled out as a source of 
the increased chromogen excretion. The findings 
suggest a previously unrecognized phenylhy- 
drazine-reacting group among metabolites of 
Provera. 

Optimal progestational response of the endo- 
metrium usually requires "priming" with estro- 



NATIONAL CANCER INSTITUTE 



23 



gen. Hisaw, et al., however, report that large 
doses of progesterone alone can induce prolifera- 
tion in the rabbit endometrium. This conceivably 
could be related to production of estrogenic sub- 
stance by the adrenal cortex. Dr. W. W. Tullner 
has observed this progestational response in 
adrenalectomized rabbits, thus eliminating the 
necessity of an adrenal factor. 

Dr. Hertz finds that prepubertal growth and 
differentiation of the ovary proceed independently 
of pituitary stimuli, because growth and differ- 
entiation progress normally when ovarian grafts 
are transplanted to hypophysectomized rats of 
either sex. Extensive destruction of the hypo- 
thalamus fails to influence the process. Ectopic 
pituitary glands continue to produce ACTH, 
growth hormone, and gonadotropin. 

Clinical experience in inhibiting the activity of 
the adrenal cortex with amphenone or with 
o,p'-DDD has pointed to the need for better drugs 
with similar primary effects but fewer undesirable 
reactions. Administration of o,p'-DDD to dogs 
not only suppresses excretion of adrenal hormones 
or their urinary metabolites but will actually de- 
stroy the adrenal cortex in 90 days. While Dr. 
Tullner has not observed evidence of regeneration 
of the cortical tissue, the period of observation 
has not yet been adequately long to be decisive. 
Despite this drastic effect in the dog, and evidence 
for comparable qualitative effects in some human 
situations, o,p'-DDD does not influence adrenal 
cortical activity in either the rat or the monkey. 

Studies of the pharmacology of o,p'-DDD have 
been hampered by its incomplete absorption on 
oral administration, so Dr. Tullner and Dr. Hertz 
have developed a suitable vehicle for intravenous 
injection of this fat-soluble compound. Secretion 
of cortical 17-hydroxysteroids decreases markedly 
within 48 hours with concomitant profound reduc- 
tion in secretion of cortisone and aldosterone 
equivalent to the depression produced by hypo- 
physectomy. 

Dr. Tullner is now studying a number of ana- 
logs of DDD. Difluorodinitrodiphenyldichloro- 
ethane inhibits production of 17-hydroxycortico- 
steroids. Diaminodiphenylmethane has an action 
similar to amphenone but produces no anesthetic 
effects and has pressor activity. This indicates 
that the methylketone structure is not essential to 



an antiadrenal effect, but the compound is too 
toxic to warrant further investigation. 

Transplantable rat adrenal carcinoma 494 is 
being studied by Drs. H. L. Stewart and K. C. 
Snell. Biochemical studies of this neoplasm by 
Dr. D. F. Johnson (NIAMD) reveal an excess of 
steroid hormones similar to those found in the 
adrenal cortex. A substrain, 494H, derived by 
passage in hypophysectomized rats, is morpho- 
logically distinct, and its characteristic histology 
is maintained on transplantation to intact rats. 
It produces polyuria, polydypsia, degeneration of 
renal tubules, and causes hyperplasia of the mam- 
mary glands, which become filled with milky secre- 
tion in both sexes. The uterus enlarges and the 
vaginal epithelium is mucified in female rats ; the 
testes and secondary sex organs of the male atro- 
phy. Growth of this tumor in hypophysectomized 
rats, however, induces development of the second- 
ary sex organs but not of the gonads. 

Thyroid neoplasms in rats and mice represent a 
wide variety of structural and biological types. 
Dr. H. P. Morris also finds their behavior incon- 
stant with respect to changes in morphology or 
growth rate when stimulated by administration of 
thyroid hormone or pituitary thyrotropin. 

Dr. S. H. Wollman and Dr. Leonard Warren 
(NIAMD) found sialic acid, a 9'-carbon amino 
sugar, in normal thyroid gland. A specimen of 
pure thyroglobulin, provided through the cour- 
tesy of Dr. Harold Edelhoch (NIAID), con- 
tained one percent sialic acid. Hence, a broader 
study of sialic acid in relation to thyroid function 
has been initiated. The highest concentration is 
found in the thyroid glands of hypophysectomized 
rats, the lowest in glands from rats fed thiouracil ; 
thyroids from euthyroid rats have concentrations 
intermediate between those two extremes. Admin- 
istration of thiouracil reduces sialic acid concen- 
trations progressively, with a minimum being 
reached at about 9 days. This may be due to the 
known resorption of thyroglobulin caused by 
thiouracil. Since some hold that thyroglobulin 
must be changed by enzymes before thyroxin is 
liberated, Drs. Wollman and Warren have meas- 
ured the concentration of free sialic acid which 
should be liberated by the same proteolytic process 
and find it to be highest at the time when concen- 
tration of total sialic acid is declining most 



24 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



rapidly. Free sialic acid content is lower in thy- 
roids from hypophysectomized than from normal 
rats. 

These basic observations have been extended to 
a study of experimental thyroid tumors in rats by 
Dr. Wollman, and Drs. Warren and S. S. Spicer 
of NIAMD. Colloid in some of the neoplasms is 
stained by the periodic acid-Schiff (PAS) reac- 
tion. Homogenates of these tumors are viscous, 
suggesting the presence of mucins. Other thyroid 
tumors contain no PAS-positive material. Many 
of the tumors contain acidic mucins in which the 
acid group proves to be sialic acid rather than sul- 
fate. Growth of one neoplasm in which sialic acid 
can be demonstrated intracellularly is associated 
with increased levels of sialic acid in blood serum 
and urine. Some of the more functionally active 
thyroid tumors contain neutral mucins, as does the 
thyroid. Autoradiographic studies of a tumor 
with neutral mucins reveal that all follicles form 
organic iodine compounds, whereas another func- 
tional tumor containing acid mucins in the colloid 
contained some follicles in which no radioiodine 
was detected. The relation of acid mucins to thy- 
roid function is being clarified. 

RADIATION BIOLOGY 

From a precise and detailed study of the de- 
leterious effects of visible light on a strain of 
haploid yeast cells, Dr. M. M. Elkind has turned 
to investigation of X-ray damage and recovery in 
mammalian cells in culture. Initial studies have 
utilized two strains of cells from the Chinese ham- 
ster, Gricetulus griseits, propagated in tissue cul- 
tures of the type originally designed by Puck. 
This permits the growth of individual cells as es- 
sentially clonal colonies whose morphological and 
biological properties can be compared. Experi- 
ence to date includes detailed analysis of damage 
produced by a single dose or fractionated doses of 
X-rays generated at 55 KV. and delivered at the 
rate of 720 rads per minute. 

These results are especially significant : 

1. The vast majority of the surviving cells com- 
pletely repair their accumulated damage before 
their first division post-irradiation. 

2. The kinetics of recovery depend on the phys- 
iological state of the cells and/or can be caused 
to appear to undergo large oscillations depending 
on the recovery medium. These apparent oscilla- 



tions may result from combined effects of changes 
in sensitivity and repair of inactivated sites. 

3. Although there are important quantitative 
differences, log-phase cells respond similarly. 

4. A cell can undergo repeated cycles of dam- 
age and repair with no apparent attenuation of 
the repair process. 

As Dr. Elkind and Miss Harriet Sutton state 
in their paper (Nature 184: 1293-1295, October 
24, 1959) : 

"There are several contexts in which these find- 
ings are of interest. If the chromosomes are the 
X-ray sensitive sites and chromosome breaks are 
the hits leading to lethality, then some new proper- 
ties of restitution must be considered. First, res- 
titution goes to completion in surviving cells. 
Secondly, the cell's ability to restitute breaks re- 
mains unimpaired after repeated doses. In view 
of the preceding, Puck's report of a high yield of 
mutant characteristics in the progeny of cells 
surviving five to seven mean lethal doses may be 
applicable to the material he was using; may be 
evidence of a radiation-induced chromosomal 
lability which is expressed after recovery and 
during clonal growth; may imply that mutation 
production and lethality are not, in general, close- 
ly connected; or may indicate that the chromo- 
somes are not the primary sensitive sites related to 
viability. 

"Another area in which these results may apply 
is in connection with tumour therapy. Treatment 
protocols involving fractionation are common, per- 
mitting in general, ample time between treat- 
ments for considerable if not complete recov- 
ery. ... If recovery is not duly accounted for, 
the survival using fractionation can be higher 
than expected by several orders of magnitude. Of 
course, tissue recovery in a general sense has been 
recognized by radiation therapists for a long time. 
These results, however, provide a cellular basis for 
this phenomenon and lend specific direction to the 
research that should be undertaken both to take 
advantage of, as well as to control, this effect." 

Similar experiments conducted by Dr. R. Z. 
Lockhart, Jr., and Dr. Elkind indicate that HeLa 
cells respond in a manner similar to those de- 
scribed for cultured cells of the Chinese hamster, 
but the HeLa line studied is not as stable. 

Dr. E. E. Bases has exploited the availability 
of single-cell culture techniques to study the in- 



NATIONAL CANCER INSTITUTE 



25 



fluence of actinomycin D on the response of HeLa 
cells to in vitro X-irradiation. He reports en- 
hanced lethal effects of X-rays on single cells ex- 
posed to the drug just before or after irradiation 
or during clone formation. Cells are more sensi- 
tive to X-irradiation when 85 percent of the water 
in the medium is replaced by heavy water, and, 
indeed, the cells can not survive indefinitely in 
the heavy-water medium. Cells surviving pro- 
longed exposure to heavy water are abnormally 
sensitive to subsequent irradiation. 

A radioactive cobalt source is now being in- 
stalled to replace machine-generated X-rays in the 
study of radiation chemistry by Dr. C. R. Max- 
well. Use of gas chromatography permits isola- 
tion, concentration, and quantification of acetic 
acid resulting from irradiation of glycine. Like 
ammonia and formaldehyde, but unlike glyoxylic- 
acid formation, acetic-acid production depends on 
the concentration of glycine in the radiated solu- 
tion as do most organic solutes. Examination by 
mass spectrography of acetic acid formed by irra- 
diation of glycine in heavy water suggests that 
the mechanism of acetic acid proposed by Weeks 
and Garrison is only partially correct. Only one- 
third of the acetic acid contains deuterium. On 
the other hand, Dr. Maxwell has confirmed quan- 
titatively the finding by Weeks and Garrison of 
substantial yields of aspartic and diaminosuccinic 
acids. One unknown compound resulting from the 
irradiation of glycine remains to be identified. 

Information on the abstraction reactions of hy- 
drogen atoms in the radiolysis of aqueous organic 
solutions is being obtained by Dr. Peter Riesz, who 
joined Dr. Maxwell during the year. When aque- 
ous organic solutions are exposed to ionizing ra- 
diation, hydrogen is produced by two distinct 
processes. The primary molecular hydrogen yield 
arises from the recombination of H atoms in a 
series of small regions of high radical concentra- 
tion and is independent of added solute under cer- 
tain conditions. Additional hydrogen is formed 
from the reactions of H atoms which diffuse out 
of the small regions and react with the organic 
solute. Analogous processes occur when organic 
solutes dissolved in heavy water are irradiated, 
and the primary molecular yield of deuterium is 
known from studies such as those reported by 
Dainton. 



Dr. Eiesz has studied abstraction reactions of 
certain amines, amino acids, amides, peptides, and 
thiols. Deuterium atoms react with glycine, acet- 
amide, and glycyl glycine at neutral pH by ab- 
stracting from carbon but not from nitrogen. In 
strongly basic solutions, an appreciable fraction 
of deuterium atoms abstract from the amino 
groups of glycine and n-butylamine. The rate of 
hydrogen abstraction for substances such as n- 
butylthiol and cysteine is higher from sulfur than 
from carbon. In neutral solutions abstraction re- 
actions account for no more than one-half of the 
deuterium atoms produced by X-irradiation. Re- 
sults for glycylglycine are particularly interesting 
in relation to radiation chemistry of proteins, since 
the reactions of H atoms with the peptide bond 
will not produce free radicals with the odd elec- 
tron on the nitrogen of the peptide bond. 

Interest in effects of total-body X-irradiation 
continues. Dr. Falconer Smith reports that re- 
covery of immune responses in mice after two 
doses of X-ray given at varying intervals demon- 
strates relationships similar to those associated 
with lethality studies. Multiple exposures are 
cumulative in injuring the immune response even 
though no effect may be noted on the leucocyte 
count. The recovery of transplantation immunity 
of cells from DBA-strain mice in LAFi hybrids 
following sublethal irradiation and various treat- 
ments which accelerate hematopoietic recovery 
are also unrelated to recovery of the blood cells. 
Dr. W. W. Smith, Mrs. Joanne Hollcroft, and 
Prof. Jerome Cornfield (Johns Hopkins Univer- 
sity) find that treatment with cells from immature 
mouse spleens hastens recovery; treatment with 
colchicine delays it. 

Dr. W. W. Smith finds important differences 
in the protective effects against lethal total-body 
irradiation of bacterial endotoxin on the one hand, 
and colchicine and its derivatives on the other. 
Endotoxins are effective in protecting young mice 
and will protect mice five weeks old, though hema- 
topoietic recovery is delayed. They also protect 
irradiated guinea pigs. The endotoxins produce 
their effects in guinea pigs only after the radia- 
tion has been given. Colchicine and the deriva- 
tives studied so far by Dr. Smith do not protect 
guinea pigs or very young mice but are active in 
mice five weeks old. They act when given prior 



26 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



to total-body irradiation but not during the post- 
treatment period. The protective effects of both 
endotoxins and colchicine are associated with more 
rapid recovery of the bone marrow among the 
treated subjects. 

"Secondary disease" is a condition occurring in 
mice at inconstant, long intervals after exposure 
to lethal doses of X-irradiation and treatment 
with protecting doses of nonisologous bone mar- 
row. The disease, which is basically an im- 
munological phenomenon, may be fatal. Miss 
Uphoff has previously associated incompatibility 
at gene locus H-2 as a major factor in production 
of secondary disease, and now reports histoincom- 
patibility at loci H-l and H-3 to be unimportant 
in the pathogenesis of the condition. Other fac- 
tors influence the long-term survival of the lethally 
irradiated and protected mice. Miss Uphoff has 
investigated the efficacy of marrow from parent- 
strain mice in the protection of F x hybrids pro- 
duced in seven different combinations. Early 
fetal marrow is effective in protecting irradiated 
hybrids in most of the combinations studied. 
Marrow obtained from neonatal mice produces 
less severe secondary disease than is obtained with 
adult marrow. When a gene difference is present 
at H-2, only 50 percent of the recipients of fetal 
marrow survive secondary disease. 

NATURAL HISTORY OF LEUKEMAS 

Earlier annual reports have included a number 
of precise clinical and pathological studies on 
phenomena associated with the leukemic state, 
such as lesions of bone, spontaneous bleeding and 
its basis, intracranial lesions associated with hem- 
orrhage, intercurrent infection, and the im- 
munologic response to specific antigens. These 
have been achieved through the continuing studies 
of leukemia patients by clinicians and pathologists 
working together on common problems. 

Drs. E. J. Freireich and E. Frei, III, have 
recognized a syndrome due to increased intra- 
cranial pressure in 25 of 150 patients, primarily 
children with acute lymphocytic leukemia. Eight 
of the twenty-five developed their first symptoms 
while in drug-induced remissions. Studies by Dr. 
L. B. Thomas and colleagues of the Pathologic 
Anatomy Branch associate the symptoms with an 
internal communicating hydrocephalus and ex- 



tensive infiltration of the pia-arachnoid by leuke- 
mic cells which in extreme cases obliterates the 
subarachnoid space over the spinal cord and brain. 
This is not a completely new lesion, but review of 
the literature reveals a sharp increase in the oc- 
currence of the syndrome since the advent of 
chemotherapy. 

All this emphasizes the need for more intensive 
investigation of the blood-brain barrier, as ap- 
proached by Drs. D. P. Rail and C. G. Zubrod. 
This physiological or biochemical barrier develops 
early in life since it is already present in puppies 
5 to 20 hours old. Neither hypotension nor ad- 
ministration of cortisone increases the entry of 
drugs into the cerebrospinal fluid. Sulfanilic 
acid, a strong acid, fails to enter the cerebrospinal 
fluid in the dogfish at 96 hours in more than traces. 
Para-aminohippuric acid, a weak electrolyte of 
which only one part in 6,000 is dissociated at body 
pH, will achieve equilibrium in time by diffusion. 
Comparison of the ability of these two compounds 
to pass the blood-brain barrier further indicates 
that undissociated rather than dissociated drugs 
will penetrate into the cerebrospinal fluid and 
strengthens the lipoidal concept of the barrier. 

Comparison by Drs. Rail and Zubrod of the en- 
try of antipyrine, sulfadiazine, and paraamino- 
hippurate into the cerebrospinal fluid of man and 
dog shows the same basic phenomena in both spe- 
cies. The human, however, approaches equilib- 
rium at a slower rate. While methotrexate does 
not readily enter the cerebrospinal fluid, Dr. T. 
L. Loo has detected 6-mercaptopurine in cerebro- 
spinal fluid of dogs receiving intravenous injec- 
tions of the drug. The spectrophotometric identi- 
fication of the compound has been confirmed by 
paper chromatography. 

Dr. George Brecher (NIAMD) and Dr. L. R. 
Schroeder, Jr., are studying synthesis of deoxy- 
ribonucleic acids in human leukemic cells by an 
in vitro technique, utilizing tritiated thymidine 
and radioautography in such a way that no new 
cells enter the synthetic phase of the generation 
cycle and none divides during the period of study. 
They observe no correlation between deoxyribo- 
nucleic acid synthetic activity and the usual mor- 
phological criteria of cellular immaturity. Al- 
though a wide range of rates of synthesis is 
observed, they do not exceed the range exhibited 



NATIONAL CANCER INSTITUTE 



27 



by leucocytes from patients with infectious mono- 
nucleosis. 

The Rebuck skin-window technique is being 
employed by Dr. Frei in a study of experimental 
inflammation in patients with acute leukemia. 
Frequently these patients fail to mobilize poly- 
morphonuclear leucocytes in response to injury. 
This abnormal response correlates well with in- 
creased susceptibility to infection and in some de- 
gree is related to the number of polymorpho- 
nuclear leucocytes in the circulating blood. 

Drs. Frei and Fahey report frequent and pro- 
gressively intense hypogammaglobulinemia in pa- 
tients with chronic lymphocytic leukemia. This 
disease is accompanied by depression of the pa- 
tient's formation of antibodies to specific antigen- 
ic stimulation. The failure of antibody produc- 
tion seems to relate more closely to increased sus- 
ceptibility of infection than to the concentration 
of y globulins in the serum. Dr. J. P. Utz 
(NIAID) ) has collaborated most effectively in 
the immunological studies. 

Dr. Frei continually reviews the infections oc- 
curring among leukemic patients. Pseudomonas 
sepsis and clostridial infections continue to be 
troublesome. A prospective study of moniliasis 
reveals thrush in 17 percent of 148 patients. Al- 
most all of the episodes have commenced during 
the last 2 months of life and duration of the epi- 
sode has been significantly diminished by a nys- 
tatin mouth wash. While antibiotic and corti- 
costeroid therapy contributes to the leukemic pa- 
tient's susceptibility to moniliasis, they are rela- 
tively less important than the severity of the 
leukemia. Disseminated cryptococcus infection 
with extensive hepatic involvement has been seen 
in three lymphoma patients. 

Drs. D. M. Watkin and I. B. Weinstein report 
that the seromucoid fraction of human plasma 
binds vitamin B 12 . Anion-exchange cellulose 
chromatography permits further separation of the 
seromucoid into three fractions. There is a 
marked increase in one of these fractions among 
patients with chronic myelocytic leukemia. 

ANEMIA AND RELATED DISORDERS 

A patient with cerebellar hemangioblastoma 
and polycythemia admitted during the year was 
studied by Dr. T. A. Waldmann, who described 

556044 — GO 4. 



marked stimulation of erythropoiesis in the rat 
by a nondialyzable factor obtained from the con- 
tents of the cystic portion of the neoplasm. This 
appears to be the first demonstration of erythro- 
poietinlike activity in any biologic material other 
than urine or plasma and supports the concept 
that polycythemia, not infrequently associated 
with cerebellar hemangioblastoma, may be caused 
by production of an erythropoietinlike factor by 
the neoplasm. 

Dr. R. E. Greenfield, Jr., has extended his 
studies of erythrocyte fragility in relation to age 
of the red-blood-cell population. Erythrocytes 
more than 12 days old hemolyze at 0.4 percent so- 
dium chloride while younger forms which he- 
molyze at a concentration of 0.2 percent can be 
further separated by exposure to gradients of salt 
content approaching 0.3 percent. The absolute 
values vary somewhat among species. Systematic 
study of red-blood-cell repletion in rats rendered 
anemic by repeated bleeding reveals fragility 
values for immature red-blood cells and reticulo- 
cytes of 0.1 percent, and 3-day-old erythrocytes of 
0.2 percent, while hemolysis occurs at 0.3-0.4 per- 
cent concentration when the red blood cells are 12 
days old. 

Dr. Greenfield and Dr. V. E. Price have also 
shown the slow disappearance from the site of dep- 
osition of erythrocytes labeled with radioactive 
iron when injected into a transplantable tumor or 
into subcutaneous tissue. The results are com- 
parable to those reported last year for studies of 
labeled erythrocytes injected into skeletal muscle. 

Having previously described the loss of catalase 
activity by parenteral injection of 3-amino-l,2,4- 
triazole which, nevertheless, does not interfere 
with synthesis of the enzyme, Drs. V. E. Price and 
Miloslav Rechcigl (Research Fellow) report ki- 
netic studies, showing that, although the rate of 
catalase destruction by aminotriazole is the same 
in both liver and kidney, the rate of enzyme syn- 
thesis is fourfold greater in the liver. Eventually 
an equilibrium is attained at which destruction 
balances replacement. 

Dr. Waldmann is attempting to produce hyper- 
splenism in dogs by intravenous infusion of meth- 
ylcellulose. Moderate splenomegaly is obtained 
together with an anemia in which life span of the 
erythrocytes is reduced. The picture is compli- 
cated by severe renal damage and uremia. Ad- 



28 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ministration of large doses of desiccated thyroid 
increases the dog's blood volume as much as 25 
percent. Increased synthesis of erythrocytes can 
be demonstrated, but their life span remains with- 
in the normal range. 

WEIGHT LOSS 

An increased requirement for sodium ions was 
reported last year by Dr. J. White as characteris- 
tic of rats bearing progressively growing Walker 
carcinosarcoma 256. He, with Dr. F. K. Millar 
and Mrs. J. N. Toal, has studied sodium metabo- 
lism in normal and cancerous rats by balance tech- 
niques. The normal subject excretes sodium in 
relation to the amount provided in the diet, but 
the cancerous rat reduces its sodium excretion 15 
to 20 days after the Walker tumor has been im- 
planted even though the animal is storing nitro- 
gen. Chloride and water follow the same pattern 
as sodium, but potassium metabolism is not af- 
fected. When the diet contains adequate amounts 
of salt, and this in considerable excess over the 
requirement of the normal rat, the cancerous sub- 
jects lose little weight despite their large tumors 
and the adrenal glands do not enlarge. The 
cachectic tumor-bearing rats whose sodium intake 
is restricted have marked enlargement of the 
adrenals affecting particularly the zona glomeru- 
losa. In rats eating a diet in which lyophilized 
tumor forms the only source of protein, the excre- 
tion of allantoin is inversely related to the excre- 
tion of sodium, though such a diet is rich in salt 
and nucleic acids. 

Dr. White and colleagues have also studied so- 
dium excretion of rats bearing the Murphy-Sturm 
lymphoma. The diet with restricted salt content 
results in initial fall in sodium excretion as the 
neoplasm grows, but the level then rises and is 
maintained as in normal rats, and the adrenals do 
not enlarge. Some tumors may have so great an 
extracellular space that the ordinary diet suffi- 
cient for growth of the normal rat cannot fulfill 
the needs of a greatly expanded extracellular 
space. It seems quite likely that a need for addi- 
tional building blocks to manufacture proteins or 
nucleic acids for a rapidly growing protoplasmic 
mass might have equally deleterious consequences 
on the host. This general subject will be explored 
further. 



At any rate, the characteristic growth pattern 
of the Walker tumor and similar consequences 
have been described in modified form for rat 
lymphosarcoma E2788 and rat hepatoma 3683 by 
Drs. Rechcigl and Greenfield. 

Dr. Pratt finds it possible to determine the gross 
body composition of cancerous rats in vivo through 
serial calculations of ratios of nitrogen stored or 
lost to caloric expenditure. Preliminary data 
indicate severe gross changes as would be antici- 
pated. The direct calorimeter for the rat, 
designed by Mr. W. C. White, has required addi- 
tional modification, but should be in use in the 
near future. 

Measurement of changes in body composition 
seems a reasonable approach to the study of 
cachexia in cancerous patients and may lead to 
some means of describing the changing mass of a 
tumor which cannot be seen or measured by more 
conventional means. Additional experience in 
the measurement of changes in body fat in the 
Siri apparatus by Drs. Berlin and Watkin, cor- 
related with metabolic balance techniques and in- 
dependent measurement of body water, reveals ex- 
cellent agreement among the values obtained by 
the different methods. 

Dr. Watkin has characterized a "weight-loss 
syndrome," which includes negative caloric bal- 
ance, low respiratory quotient, and increase in 
unesterified-plasma fatty acids, which he associ- 
ates with actively growing cancers. Study of the 
effect of fasting reveals that the patient with 
rapidly advancing malignancy either exhibits this 
syndrome at the beginning of the experiment or 
changes to that general pattern within 12 hours. 
Such persons whose basal metabolic rate is high 
initially, continue to manifest the same high rate 
despite fasting. Normal individuals, on the other 
hand, show little evidence of a "weight-loss syn- 
drome" until 16 to 36 hours after the fast begins. 

TISSUE CULTURE 

Measurement of several important biochemical 
qualities by Dr. B. B. Westfall points again to the 
high degree of chemical variation among cells 
grown in vitro for extended periods. All of the 
cell strains studied form a-keto acids. Concentra- 
tions of nucleic acids, glycogen, lipids, and cho- 
lesterol vary widely from strain to strain. No 



NATIONAL CANCER INSTITUTE 



29 



glycogen can be demonstrated in cultured fibro- 
blasts; yet after 7 years in vitro a line of skin 
epithelium stores glycogen, as do two strains of 
hepatic parenchymal cells; cells of a hepatoma 
strain derived from one of these hepatic paren- 
chymal strains, on the other hand, have lost that 
capacity. In certain cell strains, enzyme activities 
have been lost ; in one cell strain a very great in- 
crease in arginase activity was demonstrated. 
Certainly cells in rapid proliferation over long 
term in tissue culture show numerous divergent 
variations. 

A strain of monkey kidney cells obtained origi- 
nally from Eli Lilly Research Laboratories has 
been adapted to grow in a chemically defined 
medium by Dr. V. J. Evans. This strain supports 
growth of poliomyelitis virus very well. Dr. 
Evans has adapted a continuously cultured line of 
human skin epithelium to growth in chemically 
defined medium by slowly reducing the concentra- 
tion of serum in the original mixture. A new 
medium NCTC 117, derived from the familiar 
NCTC 109, omits coenzymes, sodium glucuronate, 
deoxyguanosine, deoxyadenosine, 5-methylcyto- 
sine and "essential" unsaturated fatty acids. The 
only nucleic acid derivatives required by strain 
L are thymidine and deoxycytidine. 

Dr. Sanford, studying the effects of vitamin re- 
quirements of cells growing in the chemically de- 
fined medium NCTC 117, reports pantothenate, 
choline chloride, niacinamide, thiamin, and ribo- 
flavin essential for cell survival. Folic acid, 
pyridoxal or pyridoxine, and possibly biotin, 
while not essential for cell survival, increase the 
rate of cell proliferation. 

The effect of methylcellulose in improving cell 
growth in a chemically defined medium in agi- 
tated fluid suspension cultures is not related to its 
viscosity. Mr. J. C. Bryant, Dr. Evans, Mr. E. L. 
Schilling, and Dr. W. R. Earle find no notable 
growth-promoting effect, in such cultures, of wide- 
ly different concentrations of methylcellulose on 
cells of a monkey kidney strain. 

Study of the characteristics of cell populations 
growing in vitro is made possible by comparative 
time-lapse cinematography. The first study by 
Mr. W. T. McQuilkin and Dr. Earle concerns 
changes during adaptation of one clone of strain 
L to growth in a protein-free medium. The aver- 
age generation time is prolonged and the migra- 



tion rate on the glass surface is greatly decreased, 
as is the cytokinetic phase of growth. The cells 
are less compact, refractile, and granular. They 
round up only for prophase. Monkey kidney cells, 
studied under the same conditions, demonstrate a 
generation time similar to that of fibroblasts, and 
the population increases 24-fold in a week. 

Lymphoma P388 grows well on Eagle's medium 
plus 5-percent calf serum. Dialysis of the serum 
destroys its capacity to support growth of these 
tumor cells when added to Eagle's mixture. Dr. 
Robert Roosa (Research Fellow) finds that addi- 
tion of pyruvate or Z-serine to the dialyzed serum 
restores its growth promoting properties. 

CYTOCHEMISTRY AND HISTOCHEMISTRY 

A number of chemicals cause blebbing of cells 
under in vitro conditions. Dr. M. K. Belkin has 
extended his study of blebbing to the extent that 
he considers this to be a common property of 
neoplastic cells. Experience with cells from five 
normal tissues fails to reveal bleb formation by 
the same agents that produce the change so readily 
in cancer cells. Most compounds producing blebs 
form mercaptide linkages, but a few oxidizing and 
alkylating agents also display the same property. 

Dr. Robert Love can recognize nine morpho- 
logically distinct forms of ribonucleoprotein in 
cells stained by his toluidine blue molybdate tech- 
nique. Electron-dense molybdate is deposited at 
sites of metachromasia. Some hepatic ribonucleo- 
proteins are affected by starvation; some disap- 
pear altogether and reappear on feeding. Ribo- 
nuclease in concentrations of 40 mg. per ml. has 
no effect on the staining capacities of cells. 

Dr. Love can demonstrate a nucleolinus in a 
number of normal and neoplastic cells. Although 
it is usually large in cancerous tissues, a variety of 
other unrelated conditions may also cause it to 
enlarge. Availability of the toluidine blue mo- 
lybdate technique has permitted a study by Dr. 
Love of the effect of colchicine on ribonucleic 
acids in cells. Metaphase arrest, characteristic of 
colchicine action, is associated with failure of 
nuclear parachromatin to diffuse into the spindle 
zone at the end of prophase. Some doses of the 
drug result in complete inhibition of mitosis with 
increase of parachromatin during interphase and 
enlargement of the nucleus and nucleolinus. 



30 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Parachromatin may play some role in the origin 
of spindle fibers. 

The variation in the reaction of ascites tumor 
cells to tetrazolium salts varies so widely that Dr. 
G. Z. Williams has turned his attention to study 
of mouse and rat hepatic cells, which he has 
learned to separate and to count electronically. 
Isolated mitochondria possess a variety of dehy- 
drogenases demonstrable by tetrazolium tech- 
niques, but mitochondria in whole cells react quite 
differently, as Dr. MacCardle has been saying for 
years. The differences in reaction rates observed 
in whole cells, as compared with isolated mito- 
chondria, are probably not due to differences in 
permeability, because addition of a rapid reduc- 
ing agent after 15 to 30 minutes of exposure of 
intact cells to one of several tetrazolium salts 
causes rapid reduction of the intracellular dyes. 

Dr. Williams has calculated reduction rates for 
several tetrazolium-substrate combinations. Con- 
centrations of 0.0012 M tetrazolium and 0.034 M 
succinate result in rapid, intense reduction to 
formazan in cytoplasm of hepatic parenchymal 
cells near the mitochondria with general increase 
in cytoplasmic density. Lower concentrations 
lead to accumulation of formazan in lipid drop- 
lets. 

DERMATOLOGY 

Dr. E. J. Van Scott reports that autotrans- 
plantation of epidermal tissue free of stroma from 
the donor site is either unsuccessful or the trans- 
planted cells grow and assume the characteristics 
of the epithelium of the new site. The cells will 
not grow in the dermis. Cells from a basal-cell 
carcinoma will grow in a new site only if donor 
stroma is available in the transplanted fragment. 

His continuing studies of psoriasis include the 
description of a number of agents which cause 
prompt clearing of the local lesions on topical 
application with recurrence in one to several 
weeks. Such drugs as methotrexate, 5-fluoroura- 
cil, actinomycin D and colchicine, all active 
against some neoplasms, will clear up the psoriatic 
lesions when administered systemically. Anti- 
metabolites are inactive when applied locally. 
Serial histological studies of the lesions have indi- 
cated that psoriasis is basically a hyperplastic 



process, and that cytotoxic agents retard its de- 
velopment and permit the cells to form keratin. 

Nine protein fractions, three fractions each iso- 
lated by identical methods from human epidermis, 
psoriasis scale, and ichthyosis scale, have been 
shown by Dr. Simon Rothberg to display major 
similarities when their peptide patterns obtained 
by enzymatic hydrolysis are compared. Solubili- 
zation of the proteins does not require cleavage 
of disulfide bonds. Some peptide differences have 
been observed among proteins obtained from 
normal epidermis, psoriasis, and ichthyosis. 

Additional work on the relation of hair growth 
to the dermal papilla is reported by Dr. Van Scott 
and Dr. R. G. Crounse (Research Fellow). The 
mitotic activity of the germinal matrix is propor- 
tional to the number of cells in the papilla, nor- 
mally 1 mitosis per eight papilla cells. Mitotic 
activity decreases in successively higher levels of 
the hair bulb but is a function of the area of the 
papilla to which the matrix cells are exposed and 
the transverse thickness of the matrix at each 
level. Permanent baldness results from destruc- 
tion of the papilla. Drs. Crounse and Rothberg 
find arginase in the hair sheaths but not in the 
bulbar portion of epilated roots. This suggests 
that arginase is not directly concerned with syn- 
thesis of keratin of the hair shaft. While arginase 
is known to exist in the epidermis, other enzymes 
of the Krebs-Henseleit urea cycle can not be dem- 
onstrated in this tissue. 

Detailed study by Drs. M. K. Barrett and E. J. 
Breyere (Research Fellow) of tumor transplant- 
ability among several combinations of instrain 
and outcross matings provides some unexpected 
results. Among females of a given or first strain 
of mice, those impregnated by a male of a differ- 
ent or second strain exhibit tolerance towards a 
tumor originating in the second strain. This is 
exhibited by successful transplantations of the 
neoplasm in immunized females mated to males 
of the strain from which the tumor originated, but 
not in those mated to other males. The effect in- 
creases with increasing multiparity. 

Dr. O'Gara is transplanting thymic tissues of 
newborn mice to the spleens of adult mice. The 
thymus regenerates in this location. Adrenalec- 
tomy and orchiectomy enhance the growth of the 
transplants, but ovariectomy and thymectomy pro- 
duce no effect. 



NATIONAL CANCER INSTITUTE 



31 



Some cancers clear glucose from the blood quite 
rapidly. Dr. H. A. Kahler, studying the effects of 
glucose administration on pH of an experimental 
neoplasm, has used both intraperitoneal and intra- 
venous routes of administration. The former 
causes flow of fluid from the blood into the peri- 
toneal cavity with dehydration of the tissues ; the 
latter increases the blood volume including blood 
flow through the tumor, thus producing a more 
rapid drop in pH of the neoplasm and a more 
rapid return to normal values. The reduction in 
pH following intravenous injection of glucose is 
0.65 pH in the viable part of the neoplasm, but 
only 0.16 pH in its necrotic center. 

Dr. E. D. McLaughlin has standardized study 
of the rat liver regenerating after partial hepatec- 
tomy so that reproducibility is adequate to permit 
quantification of some aspects of the process. 
Preliminary results reported by Dr. McLaughlin 
suggest that normal human serum contains some- 
thing that retards orderly regeneration, whereas 
sera from cancer patients have no such effect. 

Surgical and Virus Treatment of Cancer 

Independent measurements of circulating 
plasma and red-blood-cell volume by modern iso- 
topic techniques have been applied to study of the 
postoperative state by Dr. A. S. Ketcham and 
other members of the Surgery Branch. Once more 
the unreliability of the hemoglobin, hematocrit, 
and erythrocyte enumeration, as determined by 
conventional methods, to reflect changes in the ef- 
fective circulating blood volume is emphasized. 
During the first 2 weeks after a major surgical op- 
eration, a patient may lose 25 percent of his vol- 
ume of circulating erythrocytes without any re- 
markable change in hemoglobin concentration or 
in hematocrit. Tachycardia occurs when the effec- 
tive circulating volume of red blood cells is re- 
duced by 30 to 40 percent, and these patients re- 
spond dramatically to transfusion of two units of 
blood. 

Review of surgical experience at the National 
Cancer Institute includes 245 major operations 
and 214 minor surgical procedures. Only seven 
patients have died within 30 days of operation. 
There have been 44 postoperative infections of 
which 20 have been associated with avascular op- 
erative wounds caused either by extensive X-ir- 



radiation of the site at varying periods before sur- 
gery or by the creation of large skin flaps as part 
of the operative procedure. Antibiotic-resistant, 
coagulase-positive, hemolytic staphylococci have 
been major factors in 29 of the 44 cases, but an ad- 
ditional 17 patients known to harbor such organ- 
isms have not developed wound infections post- 
operatively. Nineteen of the forty-four patients 
had contaminated wounds that involved lesions of 
the mouth, pharynx, trachea, or the gastrointesti- 
nal tract before any operation was performed. 
This experience led Dr. R. R. Smith some time ago 
to design a prospective study of postoperative in- 
fection, and it is already clear that 23 of 30 pa- 
tients harbored, at the time of operation, the or- 
ganisms which subsequently caused postoperative 
infection. 

Continuing study by Drs. Smith, J. F. Potter, 
and Malmgren reveals that the contamination of 
wounds by cancer cells occurs most often after 
operations that involve excision of a primary can- 
cer. Neck dissection for metastatic cancer alone 
seldom results in the recovery of recognizable can- 
cer cells from the wound washings. The frequency 
of contaminated wounds in operations for epider- 
moid carcinoma continues at 26 percent and no 
correlation between contamination and local recur- 
rence is possible. Use of dilute solutions of form- 
aldehyde has been ineffective in reducing the 
frequency of local recurrences. No new leads have 
developed in the experimental study conducted by 
Dr. Ketcham in a search for agents which may kill 
the residual cells. Of the several chemicals tried, 
all promote the growth of cells when the wound is 
treated before introduction of the brie, and thio- 
tepa has a similar though less pronounced effect 
when given by intraperitoneal injection. Any one 
of the agents studied will kill cells of experimental 
tumors when suspended in the same concentration 
used to wash the wound. The wound is simply a 
different and more complex environment. 

Cancers of the head and neck are commonly 
said to metastasize to the organs and tissues below 
the clavicles in only 12 or 13 percent of patients. 
Quite a different picture is afforded from limited 
experience with this type of malignant disease 
at the National Cancer Institute. Dr. Smith has 
reviewed the 25 post-mortem examinations per- 
formed by the Pathologic Anatomy Branch on 
patients who have succumbed to cancers originat- 



32 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ing in 12 different sites of the head and neck. Al- 
most all of the lesions were epidermoid carcinomas, 
and one-half of them had not metastasized at the 
time of operation. Thirteen, a little more than 
half, of these 25 patients had distant metastases 
involving 17 different locations, and in 12 cases the 
lungs were involved. Three of the patients with 
disseminated disease never had metastases in the 
cervical lymph nodes. The average interval be- 
tween operation and death in this small group is 
only 10 months for those patients who had no 
clinically demonstrable evidence of metastases at 
the time of operation and 19 months for those 
whose cancers had spread beyond the site of origin 
when surgery was performed. Each of the latter 
series of patients was treated at least once for local 
recurrence. The prolongation of life among these 
people can hardly be attributed to the surgeon's 
skill, since they would seem to represent a some- 
what more indolent type of cancer than that which 
characterized the patients whose life expectancy 
was materially shorter. 

Urinary diversion via an ideal conduit in radi- 
cal pelvic exenteration for cancer of the uterus 
continues to give satisfactory results. Infection 
of the upper urinary tract and hyperchloremic 
acidosis are distinctly unusual in this group of 
patients, as contrasted with their frequency in 
patients with wet colostomies. Dr. Smith and 
colleagues observe a ureteral reflux when radio- 
opaque material is introduced into the ileal pouch, 
and bacterial contamination of the ureters is rela- 
tively common. 

VIRUS TREATMENT 

The use of a strain of Coxsackie virus B 3 , trained 
by Suskind, et cd, to destroy HeLa cell tumors 
growing in rats, in the treatment of advanced hu- 
man epidermoid carcinomas of the cervix (Annual 
Report, 1957) , has been studied for several years. 
The enterprise has involved many people, includ- 
ing Drs. R. R. Smith, R. B. Couch, Manaker and 
Love, NCI, and Drs. R. J. Huebner and W. P. 
Rowe (NIAID). Although this smoldering ef- 
fort is not likely to break into flame at this time, 
experience with 25 patients is interesting. 

1. Among nine patients whose serum contained 
no antibodies against Coxsackie B 3 no difficulty 
was experienced in recovering virus during the 



first 4 days after its injection per vaginam into the 
cancerous mass. Titers dropped precipitously on 
days 4 through 8 as did ability to recover virus. 
Virus was recovered from only three patients after 
the 8th day and persisted beyond the 11th day in 
only one case. Despite this experience, the total 
value calculated for recovered virus exceeds the 
amount injected originally. Among six patients 
whose sera contained antibodies against the virus, 
recovery of Coxsackie B 3 was impossible beyond 
the first 5 days following injection. 

Cultures taken from the throat and anus were 
intermittently positive for the virus up to day 6 
in some patients. 

2. Antibody titers were increased or antibodies 
appeared in all patients by the fourth day. Sub- 
sequently the titers rose to between 256 and 2,048 
about 2 weeks after treatment. 

3. Fever was the only sign of infection observed 
in 12 of the 25 patients. It exceeded 39° C. in two 
of them and occurred 12 to 72 hours after injec- 
tion. Five of seventeen patients had leukopenia 
and relative lymphocytosis. 

Injection of both adeno and Coxsackie viruses 
into the cancers of four patients produced gen- 
eral malaise and fever of 39-40° C. in 12 hours; 
the fever slowly returned to normal. All blood 
cultures were negative for bacteria. 

4. The oncolytic response was not striking, 
though a significant change was observed twice as 
frequently in the nonimmune patients as in those 
whose sera contained antibodies before treatment 
was given. No specific cytologic or histologic 
changes were observed. 

5. Tumor-to-tumor passage in patients did not 
enhance the oncolytic effect of Coxsackie virus B 3 . 

Dr. Love's studies of Newcastle disease virus 
adapted to infect and destroy Ehrlich ascites 
tumors in mice have also yielded important re- 
sults. This strain of the virus grows poorly in 
vivo in cells of the Krebs ascites tumor and origi- 
nally produced no oncolysis. Dr. Love was able 
to adapt the virus to grow in this tumor after 18 
passages in vivo, but he could not adapt original 
chick-embryo Newcastle disease virus to the 
Krebs carcinoma by serial passage in vivo. The 
original chick-embryo strain produced some on- 
colysis of lymphoma P388 ascites tumor, but the 
Ehrlich adapted strain had no such effect. Dr. 
Love concludes that adaptation of a virus to a 



NATIONAL CANCER INSTITUTE 



33 



given tumor does not confer oncolytic power 
against other neoplasms. 



deficiency, disappear when dietary desoxypyri- 
doxine is replaced by vitamin B 6 . 



PYRIDOXINE DEFICIENCY 

Keports by others indicate that pyridoxine de- 
ficiency interferes with immune responses, and 
Drs. Couch and Smith conceived the idea of en- 
hancing the oncolytic effect of adeno or Coxsackie 
B 3 viruses on epidermoid carcinoma of the cervix 
by clinically induced pyridoxine deficiency. This 
was accomplished by formulation of a semisyn- 
thetic diet which included the antimetabolite des- 
oxypyridoxine. Reports of clinical pyridoxine 
deficiency induced at other research centers 
guided the local study and, in general, each of the 
four patients studied at the National Cancer In- 
stitute pursued courses consistent with the syn- 
drome described by others. The mental symptoms 
were different in that mood swings with periods 
of stability and orientation were more frequent 
than expected. Only two patients exhibited 
marked lymphopenia. Marked impairment of 
renal function, elevated fasting blood sugar, and 
profound catabolic responses seen in each of the 
patients are new findings. Perhaps the hypergly- 
cemia may be associated with the known toxicity 
of xanthurenic acid for the /3 cells of the islets of 
Langerhans. 

The experience is too small to provide definitive 
information on enhancement of oncolysis by Cox- 
sackie B 3 virus by pyridoxine deficiency. Al- 
though signs and symptoms of the clinical de- 
ficiency were reversed promptly by replacement 
of pyridoxine for the desoxypyridoxine in the 
diet, more information on some of the previously 
undescribed effects of pyridoxine deficiency 
should be sought from animal experimentation 
before further clinical studies are undertaken. 

Drs. Dyer and Morris, having extensive ex- 
perience with pyridoxine deficiency in rats, have 
studied biochemical aspects of the clinical syn- 
drome. The urinary excretion of several trypto- 
phan metabolites is almost identical in man and 
rat during pyridoxine deficiency. The urine con- 
tains large quantities of xanthurenic and kyn- 
urenic acids, as well as large amounts of 3-hy- 
droxykynurenin and its acetyl-glucuronide and 
ortho-sulfate derivatives. These compounds, ab- 
sent from the urine before induction of pyridoxine 



Radiological Treatment of Cancers 

Diversion of the urinary stream into a surgi- 
cally created ileal pouch and X-irradiation of the 
pelvis in patients with cancers of the urinary 
bladder, class C or D, grade 3 or 4, provide marked 
symptomatic improvement in the limited experi- 
ence of Drs. J. R. Andrews, Jack Levin, and 
H. D. Suit. Temporary control of the local tumors 
has been achieved, but 9 of the 11 patients studied 
have already died of distant metastases. The two 
patients still living have survived 25 and 34 
months respectively. 

Renal function among the patients with carci- 
nomas of the urinary bladder whose ureters have 
been anastomosed to an ileal pouch has been im- 
proved, as shown by studies of blood-urea nitro- 
gen, serum creatinine, and intravenous pyelo- 
grams. Endogenous creatinine clearance and 
fractional phenosulphonphthalein excretion have 
not improved to the same extent and, indeed, seem 
to become further depressed. Bacterial contami- 
nation of the urine is unaffected by the ureteral 
transplantation, even though the patients reveal 
no clear evidence of acute pyelonephritis. 

Forty patients with cancers of the head and 
neck have been treated with protracted courses 
of 2-m.e.v. X-ray therapy. At least 1 year has 
elapsed since treatment was commenced in each 
case. The median survival is 10 months. Reac- 
tions of the skin and other normal tissues have 
been mild, probably as the result of 2-m.e.v. 
beams, rather than any increase in the time-dose 
relationship. The results are being analyzed fur- 
ther by Dr. J. R. Andrews. 

A total of 184 courses of radiation therapy has 
been given during the year by Dr. K. C. Brace 
and associates in the service treatment of many 
types of cancer. One hundred sixty of the courses 
have utilized 2-m.e.v. therapy and five involved 
treatment with electron beam. 

Mr. R. W. Swain is replacing the dosimeters 
used in conjunction with 2-m.e.v. Van de Graaff 
electrostatic generators with transmission ioniza- 
tion chambers connected to current integrators 
which can be preset to turn off the machine after 



34 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the prescribed dose has been given. Mr. Swain 
has also devised a means to permit semiautomatic 
plotting of isodose distribution in water using 
small ionization chambers. 

Drs. J. E. Andrews, R. L. Swarm and associates 
have studied another patient with extensive chon- 
drosarcoma of the thoracic wall whose neoplasm 
concentrated radioactive sulfur. A therapeutic 
dose of 388 mc of radioactive sulfate was followed 
after 2 months by another dose of 335 mc. Tem- 
porary retardation of tumor growth was followed 
later by increase in growth, requiring external 
beam-irradiation therapy. The isotope treatment 
produced only slight depression of leucocytes and 
platelets until 4 weeks after administration of the 
second dose, when thrombocytopenia became 
marked and leukopenia moderate. The blood pic- 
ture returned to normal in 2 months, but 6 months 
after the second dose of isotope the patient de- 
veloped a severe anemia and thrombocytopenia. 
Another patient treated in much the same way 
more than a year ago has developed no such syn- 
drome which recalls experience with dogs reported 
by Dr. Brecher some years ago. 

Dr. D. P. Tschudy has studied metabolic effects 
of X-irradiation in a patient with a lymphosar- 
coma of the lower extremity. Prior to treatment, 
the metabolic pool of nitrogen was slightly in- 
creased, but no change in the rate constant of 
metabolic pool turnover could be detected. A 
dose of X-rays amounting to 1,000 r was followed 
by marked enlargement of the pool with decrease 
in the same constant. Incorporation into body 
protein of amino acids labeled with N 15 , however, 
was unchanged during this period. 

In the experimental laboratory Dr. Suit has 
studied the influence of increased oxygen tension 
on the response of transplantable adenocarcinoma 
in C3H/BA to local X-irradiation. He reports no 
enhancement of therapeutic effect of the X-ray 
treatment when mice bearing this neoplasm are 
exposed to an environment of pure oxygen at 2 
atmospheres during X-irradiation, as compared 
to an environment of air at 1 atmosphere. The 
result is the same with tumors measuring either 
1 or 2 centimeters in diameter. A statistically 
significant difference in radiation effect was ob- 
tained with the smaller neoplasms. 

The resignations during the year of Mrs. Holl- 
croft and Dr. Suit require complete rebuilding of 



the laboratory component of programs relating to 
therapeutic use of ionizing radiations. Difficulty 
in recruiting suitable patients for clinical radio- 
logical research has also retarded progress. 

Chemotherapy of Cancers 

Critical analysis of experience obtained 
throughout the world in the drug treatment of 
cancers reveals a small group of specific anatom- 
ical types of neoplastic diseases in which chemo- 
therapeutic agents have produced definite bene- 
ficial effects, and a much larger group of cancers 
which remain relatively unaffected by systemic 
administration by chemotherapeutic agents. 
There are, therefore, at least two separable, broad, 
general problems with an infinite variety of spe- 
cific subordinate problems. The substantial prog- 
ress made in treatment of leukemias, lymphomas, 
choriocarcinoma, and adrenal cortical neoplasms 
emphasizes the desirability of quantitative com- 
parisons of the efficacy of chemotherapeutic agents 
already available, and further exploration of 
chemical relatives of the effective agents for 
greatly enhanced therapeutic activity and research 
designed to elucidate the most effective means of 
administering the drugs now on hand. A search 
for new drugs with qualitatively different types 
of activity may also benefit patients with respon- 
sive types of cancer, but this becomes of para- 
mount importance to patients suffering from the 
much larger number of neoplastic diseases which 
respond fleet ingly or not at all to the currently 
available drugs. A variety of transplantable ani- 
mal tumors is available for this research, and 
probably the variety must be increased if the neces- 
sarily empirical approach is to be fruitful. In- 
creased emphasis on biochemical investigations 
with a view to categorizing individual or special 
types of cancers seems practical and desirable. 
Certainly a much greater use of patient material 
will be necessary before meaningful correlations 
between the therapeutic reaction of man and ex- 
perimental animals can be established and, indeed, 
we probably do not know how to study therapeu- 
tic responses of cancers of the stomach, large in- 
testine, or kidney at this time with any degree of 
precision. While the history of therapeutic re- 
search is sprinkled with episodes as dramatic as 
the penicillin story, most of the major advances 



NATIONAL CANCER INSTITUTE 



35 



have required slow exploitation of small gains by 
systematic study. 

The course of any research requiring creation 
of new knowledge is largely unpredictable. Hav- 
ing pointed out the possibilities of exploiting cer- 
tain important developments obtained from the 
study of plasmacytomas, it seems appropriate to 
recount progress in correlated clinical and labora- 
tory research in a different kind of cancer. 

CHORIOCARCINOMA 

The introduction of methotrexate therapy of 
choriocarcinoma stemmed from basic information 
on the need for folic acid in uterine and fetal me- 
tabolism, and, indeed, the initially effective thera- 
peutic regimen designed by Drs. Hertz and M. C. 
Li deviated significantly from generally accepted 
doses and schedules of antifolic compounds. 
Fifty female patients with choriocarcinoma have 
now been admitted to the National Cancer Insti- 
tute under Dr. Hertz's care. Twenty-one of the 
forty-four patients admitted prior to September 
15, 1959, are free from clinical, radiological, or 
hormonal evidence of residual disease for periods 
ranging from 5 to 47 months. Eight have per- 
sistent choriocarcinoma, and 15 have died follow- 
ing an initial response to methotrexate therapy, 
with subsequent development of methotrexate re- 
sistance. Acquired drug resistance is an impor- 
tant obstacle to more effective management of this 
neoplasm. Methotrexate-resistant patients do 
not respond to subsequent treatment with nitro- 
gen mustard, Cytoxan, or actinomycin D. 

Since May 1959, Dr. Hertz and associates have 
treated eight patients suffering from met'hotrex- 
ate-fast choriocarcinoma with Vincaleukoblas- 
tine, an alkaloid of Tinea rosea, initially de- 
scribed by Noble, Cutts, and Beer, and made 
available through courtesy of Eli Lilly and Com- 
pany. Three of these women are now in complete 
remission, the longest period being 5 months. 
Two others had definite but rapidly reversible 
evidence of tumor regression ; no response was ob- 
tained in the other three. Vincaleukoblastine de- 
presses the bone marrow, causes central nervous 
system toxicity, alopecia, stomatitis, malaise, and 
local phlebitis at the site of injection; all of these 
effects are reversible. Nevertheless, it affects the 
course of methotrexate-fast choriocarcinoma, be- 



cause it is the first alkaloid to retard cancerous 
growth. 

To obtain a better understanding of problems 
associated with choriocarcinoma, Dr. Hertz 
adapted four different strains of human chorio- 
carcinoma to grow in the cheek pouch of condi- 
tioned hamsters, but two of the strains no longer 
require pretreatment of the host with cortisone. 
Each of the tumor strains grows progressively for 
2 to 3 weeks, when central necrosis occurs, fol- 
lowed by liquefaction and absorption of the graft 
in 1 to 2 weeks. Continued administration of cor- 
tisone does not influence this picture. Hamsters 
in which the neoplasm has been absorbed will not 
grow any of the strains of choriocarcinoma, and 
passive immunity can be conferred on other ham- 
sters by injection of serum from the resistant 
animals. The resistant state is not induced by in- 
jection of several human tissues including normal 
placenta. 

The problem of the immune relationship be- 
tween choriocarcinoma and the host subject has 
been raised frequently because this neoplasm is 
derived from the zygote and contains genes from 
both parents. Dr. Hertz has undertaken a study 
with Dr. P. J. Schmidt (DBS) of the blood 
groups of choriocarcinoma patients and their hus- 
bands. No evidence of cross-immunization be- 
tween the marital partners has been found, even 
among patients whose tissues were extensively in- 
vaded by tumor. The scientists infer that the 
embryonic neoplasm lacks significant antigenicity 
for the host, at least as far as circulating antibody 
formation is concerned. 

The heterologous choriocarcinoma transplants 
produce large amounts of chorionic gonadotropin, 
which are readily detectable in the tumor, plasma, 
and urine but cannot be demonstrated in the host's 
normal organs or tissues. Choriocarcinoma also 
produces the gonadotropin in tissue culture. Dr. 
Hertz finds that neither slices nor homogenates 
of normal hamster organs inactivate the hormone. 
Unlike the normal placenta, choriocarcinoma dis- 
plays no estrogenic, progestational, corticoid, 
thyrotropic, or adrenotropic activity. 

Dr. Hertz is using the transplanted choriocar- 
cinomas in screening for chemotherapeutic activi- 
ty. Even though a particular tumor may have 
been taken originally from a patient whose lesion 
was completely resistant to methotrexate, the het- 



36 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



erologous transplant still displays moderate sensi- 
tivity to the drug. The most potent tumor in- 
hibitors found thus far are Vincaleukoblastine, 
noted above, and a related Vinca alkaloid, Leuro- 
sine. Other alkaloids of Vinca rosea have been 
inactive when given in toxic doses. 

Thus a program originating in the laboratory 
has had important consequences in the clinic 
where new and additional problems have arisen 
requiring further laboratory research at an even 
more fundamental level. It is difficult to study 
choriocarcinoma in experimental animals and 
study of the physiology of chorionic gonado- 
tropin presents problems. Serum and urine of 
the pregnant female monkey, Macacus rhesus, 
studied by Dr. Hertz, contain detectable amounts 
of chorionic gonadotropin only from day 20 to 
day 35 following timed mating. The hormone 
cannot be demonstrated in concentrates of these 
body fluids at any other time, nor is it found in 
any of the tissues including the chorion itself. 

ENDOCRINE THERAPY 

Administration of human pituitary growth hor- 
mone to 10 patients with metastatic carcinoma of 
the breast, 3 patients with cancers of the adrenal 
cortex, and 2 men with prostatic carcinoma has 
produced no exacerbation of disease nor other 
changes in the measurable parameters of tumor 
activity, according to Drs. Bergenstal and Lipsett. 
Excretion of urinary calcium is not influenced by 
injection of growth hormone during remission in- 
duced by hypophysectomy. Only those patients 
whose cancers continue to grow progressively 
after removal of the pituitary respond to growth 
hormone administration by some increase in uri- 
nary calcium excretion. 

Dr. J. M. Van Buren (NINDB) has performed 
section of the pituitary stalk in 11 patients with 
advanced mammary cancers. The morbidity has 
been greater than in patients submitted to hypo- 
physectomy, and the overall results are not as 
good. Drs. Bergenstal and Lipsett report decline 
in adrenal cortical activity among these persons 
to the levels reached in hypophysectomized indi- 
viduals. Gonadotropin excretion is variable. 
Four patients demonstrated normal thyroid ac- 
tivity which could be suppressed with triiodothy- 
ronine. The investigators regard these findings as 
evidence for a variable degree of destruction of 



the anterior pituitaiy following stalk section. Un- 
like the dog, ACTH production seems to be the 
most sensitive index of this type of pituitary dam- 
age in man. Thyroidpituitary relationships in 
several patients have remained normal in the ab- 
sence of vascular connections between the hypo- 
thalamus and pituitary. 

o,p'-ODD 

Drs. Hertz, Bergenstal, and Lipsett have used 
o,p'-DDD in the treatment of 14 patients with 
metastatic carcinoma originating in the adrenal 
cortex. All have shown some suppression of 
steroid output, and objective regressions of cancer 
have occurred in five. Ketosteroids reached low 
levels in two patients who responded to treatment, 
but a large proportion of the excretion was identi- 
fied as dehydroepiandrosterone, which suggests 
persistently active carcinoma. Undesirable side 
reactions of o,p'-DDD encourage investigation of 
related compounds for better therapeutic agents. 

SCREENING ACTIVITIES 

The contract with Microbiological Associates, 
Inc., for quantitative screening of chemotherapeu- 
tic agents continues to be a highly satisfactory 
venture, under the direction of Dr. Zubrod, Dr. 
Abraham Goldin, and Mr. J. M. Venditti. 
Seventy-eight new compounds have been tested 
for therapeutic activity against leukemia L1210, 
using methotrexate as a reference standard, and 
10 additional agents have been retested. A sub- 
stantial amount of information has been obtained 
on the effect of dosage schedules. Some agents, 
especially cytotoxic antibiotics, are more effective 
when administered daily, while other agents are 
more efficacious when given at more widely sep- 
arated intervals. Dosage schedules can influence 
therapeutic responses even among members of the 
same chemical class. Dichloroamethopterin, for 
example, exhibits about the same therapeutic effect 
when given twice daily or at intervals of 4 days 
while methotrexate is much less effective when the 
la.tter schedule is used. 

Thirty-five compounds have been tested against 
sarcoma S-37 which Dr. Goldin has adapted to 
grow reproducibly for use in the screen. Treat- 
ment is usually started 3 days after tumor implan- 
tation and continues daily for 5 days. Seven com- 
pounds produce 40-percent inhibition of growth 



NATIONAL CANCER INSTITUTE 



37 



on the 10th day after inoculation of the tumor 
when administered in doses causing death in not 
more than 20 percent of the animals. These com- 
pounds in optimal dose also increase the median 
survival time at least 40 percent over untreated 
control mice. Another five agents inhibit growth 
of S-37 without increasing survival time. S-37 
has been particularly resistant to chemotherapeu- 
tic attack, and these results reported by Dr. Goldin 
and Mr. Venditti are encouraging. 

Carcinoma 755 is the third transplantable neo- 
plasm to be introduced into the quantitative screen. 
The reference standard against which other com- 
pounds are tested is 6-mercaptopurine, which pro- 
duces 80-percent increase in survival time, and 
some tumor-free survivors are obtained when 
treatment is begun on the fourth day after inocu- 
lation and continued daily for 5 days. A large 
number of chemotherapeutic agents will retard the 
growth of this neoplasm and prolong the lives of 
the hosts when administered according to the same 
treatment schedule. Most of the more effective 
compounds are purine and pyrimidine antimetab- 
olites. 

Much of the research conducted by Drs. Dean 
Burk and M. W. Woods is directly concerned with 
the mechanism of drug action. The writer be- 
lieves, however, that the total experience should 
also be viewed as a potential means of screening 
compounds for chemotherapeutic activity. The 
techniques seem capable of using clinical as well 
as experimental cancers, even though some tech- 
nical difficulties may be encountered. Drs. Woods 
and Burk report that a high degree of malignancy 
is associated with greatly increased glycolytic 
capacity and lowered sensitivity to glycolytic in- 
hibition of the anti-insulin type (Annual Reports, 
1956-58). Generally speaking, those experimen- 
tal neoplasms in which the hexokinase reaction is 
under strong insulin anti-insulin control respond 
to one or more chemotherapeutic agents better 
than do those cancers in which glycolysis is less 
readily inhibited by compounds of the anti-insulin 
type. One does not have to accept the theory ad- 
vanced by Drs. Burk and Woods to recognize that 
these scientists have found a means of classifying 
experimental cancers with respect both to degree 
of malignancy and potential effectiveness of 
chemotherapeutic drugs. 



Drs. Burk and Woods have studied the antigly- 
colytic effect of 5-fluorinated pyrimidines against 
Krebs-2 and Ehrlich ascites tumors, finding 5- 
fluorouridine the most active and 5-fluorocyosine 
inactive. The in vitro studies correlate closely 
with the capacity of each compound to inhibit 
tumor growth in vivo. On the other hand, a con- 
centration of 400 P.P.M. of uridine is as active in 
vitro as is 20 P.P.M. of 5-fluorouridine. The anti- 
glycolytic activity of the 5-fluorinated pyrimidines 
can be largely counteracted by increasing inor- 
ganic phosphate in the incubation medium, re- 
moval of oxygen, or by the use of agents that un- 
couple phosphorylation. The effect measured by 
Warburg manometry requires glucose; succinate, 
pyruvate, or glutamate cannot be substituted for 
glucose. Experiments conducted with Mr. J. C. 
Hunter describe the inhibition of aerobic gly- 
colysis of rat-bone marrow by 5-fluorouracil, but 
not by 5-fluorodeoxyuridine and reflect the relative 
degree of toxicity of these compounds for mice. 

Drs. Woods and Burk report that the in vitro 
mechanism of action of the 5-fluorinated pyrim- 
idines appears to involve an inhibition of aerobic 
phosphorylation which lowers the availability of 
adenosinetriphosphate (ATP) for glycolysis. In- 
sulin lowers the requirement for ATP and should 
counteract the metabolic effects of the 5-fluori- 
nated pyrimidines. This it does in S91 melanoma 
and normal bone marrow, but not in insulin-in- 
sensitive ascites tumors Krebs-2 and Ehrlich. 

Differences in metabolic characteristics of tum- 
ors respectively susceptible and resistant to action 
of a given chemotherapeutic agent are reported by 
Dr. Burk and Dr. K. M. Wight. 8-Azaguanine 
produces a prompt and marked increase in gly- 
colysis in vitro of susceptible leukemic cells but 
not of resistant cells with concentrations of drug 
ranging from one-fourth to four times the pharm- 
acological level commonly used to influence the 
growth of the sensitive leukemia in vivo. Respira- 
tory inhibition occurs in both sensitive and re- 
sistant cells exposed to 8-azaguanine but is seen at 
lower doses with the susceptible line. The meta- 
bolic effects characteristic of the sensitive cells 
tend to disappear after repeated daily exposure to 
the drug in vivo and may be completely lost with- 
in a transplant generation. 

The action of Cytoxan on the metabolism of 
susceptible mouse tumors leukemia L1210, K-2 



38 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ascites, and Ehrlich ascites has also been studied 
by Drs. Burk and Wight. Treatment in vivo with 
removal of the tumor cells for study in vitro re- 
veals pronounced inhibition of aerobic and anaero- 
bic glycolysis as well as of respiration. Respira- 
tory and glycolytic functions of a cytoxan-resist- 
ant strain of L1210, produced by Dr. Montague 
Lane, were usually not inhibited by treatment and 
are sometimes stimulated. 

ANTILEUKEMIC COMPOUNDS 

Cooperative Leukemia Group B in which Drs. 
Frei and Freireich represent the National Cancer 
Institute has completed its study of the relative 
effectiveness of methotrexate and 6-mercapto- 
purine given alone or in combination for the treat- 
ment of acute leukemia. The study includes ex- 
perience with 328 patients, of whom 92 were hos- 
pitalized at Bethesda. The highest remission rate 
was attained when both drugs were given concur- 
rently, but the rate was no higher than the sum 
of the remission rates produced with either com- 
pound alone. The remission rate was the same 
for either 6-mercaptopurine or methotrexate 
whether used as primary treatment or used follow- 
ing the administration of the other drug. Treat- 
ment with 6-mercaptopurine alone, however, pro- 
duced a higher remission rate than was obtained 
with methotrexate. Overall rate of remission 
for all schedules was 50 percent of children, 15 
percent in adults, but methotrexate was almost 
inactive in acute adult leukemias producing bene- 
ficial effects in less than 5 percent of those patients. 
Toxicity, comparable in all groups, required ces- 
sation of treatment after 9 to 25 days (average 14 
days). Methotrexate produced more oral symp- 
toms, 6-mercaptopurine, more gastrointestinal dis- 
orders. 

In attempting to apply to the clinical problem 
knowledge of the biochemical mechanism involved 
in the resistance of experimental leukemias to 6- 
mercaptopurine, Dr. J. D. Davidson has encount- 
ered methodological problems. He has synthe- 
sized 6-mercaptopurine labeled with S35, but use 
of this material has necessitated development of 
new techniques for isolation of the drug's metab- 
olites. 

One problem confronting the clinical investiga- 
tor relates to procurement of a sufficient quantity 



of a promising new drug to permit definitive 
clinical trial. The staff of the CCNSC has been 
most helpful in such matters, and the Lederle Di- 
vision of the American Cyanamid Company co- 
operated to the fullest possible extent in making 
3',5' dichloroamethopterin (DCM), but it took 
such a long time that no substantial progress can be 
reported in therapeutic trials against leukemias 
or other cancers in man. Studies are now under- 
way, but the bulk of work with this new compound 
accomplished during 1959 relates to experimental 
cancer. 

Dr. Loo and Dr. V. T. Oliverio can separate 
methotrexate (MTX) from DCM by column chro- 
matography and can also extract it from body 
fluids. Only 10 percent is recovered unchanged 
from the urine after administration to man or 
mouse. Another 5 percent of the given dose is 
altered in some undetermined way. 

DCM is the drug most effective against mouse 
leukemia L1210 and, indeed, some mice are ac- 
tually cured of their advanced disease by use of 
this compound (Annual Report 1958). Dr. 
Goldin with Dr. M. A. Chirigos and Messrs. 
Venditti, S. R. Humphreys, and G. O. Chapman 
have conducted extensive experiments on DCM. 
Daily treatment must be continued 30 to 60 days 
to permit complete recovery from L1210. Ordi- 
narily the tumor disappears in about two weeks 
but recurs promptly if treatment is stopped pre- 
maturely. Oral administration of both DCM and 
MTX is less effective in experimental leukemia 
than is subcutaneous injection. 

Drs. Goldin, A. W. Schrecker, and J. A. R. 
Mead (Visiting Scientist) report that inhibition 
of formate incorporation into the acid soluble 
adenine of leukemic spleen is a reasonable meas- 
ure of the effective doses of antifolic compounds 
given by parenteral injection. Maximum effect 
occurs 20 minutes after injection of the drug but 
one hour after oral dosage, and larger doses are 
needed to produce the same quantitative effect. 

Some clue to a reason for the increased effec- 
tiveness of DCM over MTX in the management 
of experimental leukemia L1210 is afforded by 
other reports from the same investigators and Dr. 
R. A. Darrow. DCM has a smaller inhibitory 
effect on formate incorporation than does MTX. 
The dose ratio for equal response is about two, 
but the effect from MTX lasts longer. Extending 



NATIONAL CANCER INSTITUTE 



39 



treatment intervals to 6 hours or more increases 
the dose ratio of DCM to MTX to 25. A single 
dose of DCM needed to inhibit formate incorpo- 
ration for 24 hours must be of the order of 75 
mg/kg, the same dose that produces maximum 
survival time of leukemic mice. A comparable 
dose of MTX cannot be tolerated. 

Mice cured of L1210 will usually grow neither 
a second graft of this neoplasm nor cells from a 
subline of L1210, M46R, which resists treatment 
with antifolic compounds. If growth does occur, 
the new tumor remains localized to the site of 
implantation. BALB/C mice can be immunized 
against L1210. This type of immunity can be 
overcome, however, by treatment with MTX if 
the mice are inoculated with the antifolic-resist- 
ant M46R. If, on the other hand, immunity is 
induced by injections of spleen from strain DBA, 
the natural host of L1210, or by other experi- 
mental leukemias, antifolic compounds do not al- 
ter the resistant state. 

These observations raise questions concerning 
the role that immunity plays in influencing the 
host-tumor relationship in experimental chemo- 
therapy. Dr. Goldin and associates have per- 
formed an experiment in which the markedly re- 
sistant subline M46E has been affected by admin- 
istration of an antifolic compound. Mice bearing 
both the sensitive form of L1210 as well as the 
resistant form M46B, were treated with DCM. 
Their lives were prolonged for as much as 60 days, 
though treatment of M46K alone with either 
MTX or DCM extends the survival time to 20 days 
as compared with the 10-day life expectancy of 
untreated controls. Presumably the effective 
treatment of the sensitive tumor elicits an immune 
response in the host, which retards the growth of 
the resistant variant. 

Drs. Goldin, Schrecker and Mead report further 
that large doses of MTX inhibit formate incor- 
poration by both sensitive and resistant lines of 
leukemia L1210. Inhibition is less pronounced by 
treatment of the resistant cells with small doses of 
the drug. Such differences are quantitative rather 
than qualitative and are more pronounced in the 
solid tumor than in the leukemic spleen. Citro- 
vorum factor only partially reverses formate in- 
hibition induced by antifolic therapy. Perhaps 
the transport of citrovorum factor into the cells 
may be impaired by MTX administration, as was 



demonstrated for certain bacteria by Wood and 
Hitchings. 

Experiments conducted by Mr. F. G. Dhyse sug- 
gest some relationship between other vitamins and 
folic acid. When biotin is added to cultures of 
L. arabinosus in excess of the quantity required for 
maximum growth, the cultures give rise to a five- 
fold increase in folic acid in the medium without 
any increase in growth rate. The folic-acid con- 
tent of the bacterial cells is not affected, and the 
biotin must be added while the culture is growing 
actively. If excess quantities of pantothenate or 
riboflavin instead of excess biotin are added to the 
medium, excessive production of folic acid is not 
observed. 

Dr. Michael Potter is also interested in folic 
acid and its antimetabolites and uses the tetra- 
ploid lymphocytic neoplasm P288 rather than 
L1210 as a tool. A f olic-acid-deficient diet formu- 
lated by Dr. G. M. Briggs (NIAMD) produces 
better survival of mice bearing P288 resistant to 
antifolic drugs than of mice bearing the drug- 
sensitive counterpart. 

In studying the acquisition of the resistant state, 
Dr. Potter inoculates 10 cells into genetically ap- 
propriate mice and 80 percent of the recipients 
develop leukemia regardless of whether the inocu- 
lum is derived from cells resistant or sensitive to 
the action of antifolic compounds. If the sensitive 
leukemia is treated five times within 10 days with 
small doses of MTX, transplantability falls to 5 
percent. Cells isolated from P288-sensitive lines 
which have been treated with larger doses of MTX 
for longer periods of time grow in 30 to 60 percent 
of the mice into which they are transplanted. 
Furthermore, the appearance of a progressively 
growing mass at the site of inoculation is delayed 
for some time, suggesting some incomplete and 
hitherto unknown type of resistance. 

Dr. Goldin, Mr. Venditti, and Dr. Frei describe 
a series of pyrazolopyrimidines that exhibits a 
wide range of antileukemic activity against L1210. 
The most effective of these chemicals, however, is 
far less potent than either 6-mercaptopurine or 
methotrexate. 

Virus-induced leukemias have been introduced 
into the chemotherapy program by Dr. Moloney, 
Dr. Goldin, and Mr. Humphreys. No increase in 
survival time has yet been obtained with metho- 
trexate, 6-mercaptopurine, or Cytoxan, even though 



40 ANNUAL REVIEW OF INTRAMURAL RESEARCH 

definite reduction in the size of affected organs has Uracil mustard, another alkylating agent, has 

been observed. also been studied in the clinic. Drs. Brindley and 

Lane find it active against Hodgkin's disease, lym- 

AT KYT ATlNr AfFNTs; phomas, and chronic leukemias in oral weekly 

doses of 0.2 mg/kg. The drug depresses the bone 

A new alkylating agent, cyclophosphamide, de- marrow and occasionally causes severe gastroin- 

veloped in Germany, is commonly called Cytoxan. testinal symptoms. The following oral doses are 

Dr. Montague Lane reports an LD 50 of 425 mg. tolerated for 6 weeks with minimal toxicity : 

for mice, 150 mg. for rats. The drug produces „. , 

little gastrointestinal toxicity and causes hyper- _ ., . ' 1t g ,, 

, • j. , , t , J . ,, , J ^ Daily dose 0. 015 mg/kg 

plasia ol the megakaryocytes m the bone marrow „, ^ , n n n 

, , n , & . J ,. ,, ., Weekly dose 0. 2 mg/kg, 

and spleen. Cytoxan is more active than nitrogen J ° 

mustard against leukemia L1210, including sub- A comparative study of the effectiveness of 
lines resistant to antifolics, lymphoma L-2, car- thiotepa relative to nitrogen mustard in the treat- 
cinoma 241-6, and the Dunning rat leukemia and ment of Hodgkin's disease indicated superiority 
lymphosarcoma. The drug is most effective when of the latter drug (Annual Report 1958). Re- 
administered once weekly. Dr. Goldin has con- examination of the data by the Eastern Solid 
firmed Dr. Lane's finding with respect to the action Tumor Group disclosed some evidence that the 
of Cytoxan on L1210 and reports it at least as dose of nitrogen mustard was closer to the maxi- 
effective as methotrexate. Many mice survive in- mum tolerated dose than was the dose of thiotepa 
definitely when treatment is instituted early in the used in the study. Therefore, the investigators 
course of the transplanted leukemia. Cytoxan is undertook a study of dose-response relationships 
also effective against adenocarcinoma 755 and for both these alkylating agents. It soon became 
$-37. apparent that the dose-response curve is steep for 

Studies of the toxicology of Cytoxan in dogs and nitrogen mustard, since therapeutic activity is lost 

rats have been carried out under the direction of when the usual dose is reduced 50 percent. 

Dr. D. P. Rail at the Hazleton Laboratories. The Whether the therapeutic effects of thiotepa and 

advantage in terms of administration of large nitrogen mustard on Hodgkin's disease are similar 

quantities of drug through the use of widely at maximally tolerated doses remains to be seen 

spaced dose schedules was clearly borne out for at the end of the study. 

these species. Of particular interest is the demon- During the course of these several studies, Dr. 

stration in rats of a period following adminis- Brindley has followed the activity of the enzymes 

tration of large doses during which the animal ap- lactic acid dehydrogenase and ribonuclease in the 

pears to be refractory to toxic manifestations of sera of many patients. When treatment is success- 

cytoxan or methotrexate. ful, the activity of these enzymes decreases. The 

Cytoxan in man produces greater depression of activities are also likely to fall in preterminal 

leukocytes than of platelets, according to Dr. C. stages of cancer. The use of serum enzyme aotiv- 

O. Brindley and Dr. Frei. Daily oral doses of 4 ity for following the course of cancer is limited 

mg/kg are tolerated for weeks. Some therapeutic additionally because many patients never display 

activity is displayed in patients with lymphosar- any increases in lactic-acid dehydrogenase or 

coma or ovarian carcinoma. ribonuclease. 

Leukemia group B is studying the effect of Protection of bone marrow from deleterious 

Cytoxan therapy in acute leukemia. Drs. Frei- effects of X-radiation by prior administration of 

reich and Frei report no difference in toxicity 2 - aminoethylisothiuronium bromidehydrobro- 

when given daily or weekly, but somewhat more mide (AET) encouraged Dr. Kelly to study its 

therapeutic benefit appears to be obtained from effectiveness in preventing bone-marrow damage 

weekly doses. A total of 96 patients has been associated with the use of nitrogen mustard. The 

studied, of which NCI has contributed 30. Pre- Dunning-rat leukemia is highly sensitive to nitro- 

liminary results indicate objective improvement in gen-mustard therapy. No advantage was gained 

30 percent of the patients. by pretreatment with AET when the cytotoxic 



NATIONAL CANCER INSTITUTE 



41 



agent was given in small doses, but some protec- 
tion was demonstrated against toxicity produced 
by larger doses of nitrogen mustard. A series of 
new compounds which generate free radicals is 
being screened for similar protective effects by 
Drs. Kelly and Loo. 

TETRACYCLINES 

Although tetracyclines do not appear to arrest 
growth of cancer, their important antibiotic prop- 
erties and their ability to concentrate in certain 
tissues including some cancers makes desirable a 
study of the factors governing their cellular dis- 
tribution. This is being accomplished by Dr. K. 
W. Kohn, working with Drs. Loo and Rail. 
Methods for extracting the drug from plasma, 
red blood cells, spinal fluid, urine, and bile were 
developed readily. Tissues presented a different 
problem because of their relatively high phosphate 
content. This was solved by precipitating the 
phosphates with lead ions at pH 5. The method 
is highly sensitive for certain tetracyclines, but 
other members of the family cannot be extracted 
by the procedure. 

Study of plasma concentrations of tetracycline 
and dimethylchlortetracycline after single intra- 
venous doses in dogs revealed first-order disap- 
pearance curves. Calculation of volumes of dis- 
tribution indicated that dimethyl compound and, 
to a lesser extent, tetracycline are concentrated in 
some extravascular compartment of the body. 
The drugs entered the cerebrospinal fluid in con- 
centrations approximating 20 percent of those 
found in plasma, though equilibrium was not at- 
tained. Concentrations in skeletal muscle 6 hours 
after administration were higher than in the 
plasma. Considerable amounts of both drugs 
were found to be associated with erythrocytes. 
While the disappearance curves from red blood 
cells were similar to those described for plasma, 
the presence of divalent metal ions had important 
effects which probably influence distribution of 
the tetracyclines in vivo. 

Formation of complexes of tetracycline with 
calcium and other divalent metals is well-known. 
Dr. Kohn also describes complexes formed with 
barbiturates and with /?-diketones and studies of 
the influence of divalent metal ions on complex 
formation. Tetracycline forms a unionized com- 



plex with barbital and either calcium or zinc ions. 
Magnesium and manganese fail to form extract- 
able complexes. Pheno- and pentobarbitals are 
even more potent in causing extraction than bar- 
bital itself, and methyl substitution of a single 
nitrogen in the barbiturate nucleus does not 
change this property. If both nitrogens are 
methyl substituted, the molecule becomes inert. 
Such complexes do not change either the absorp- 
tion spectra or optical density of the tetracyclines 
studied, suggesting that the barbiturate may not 
bind to the chromophoric part of the molecule. 

In light of these findings, Dr. Kohn has per- 
formed equilibrium diatysis studies to determine 
whether tetracyclines bind to nucleic acids and 
proteins. The most striking results are obtained 
with deoxyribonucleic acids, which bind the drugs 
in the presence of calcium, zinc, or magnesium 
ions. Binding does not occur in the absence of di- 
valent metal ions. Heat denaturation increases 
the binding ability of deoxyribonucleic acids. 
Ribonucleic acids bind tetracyclines less well and 
albumin binds to only a small extent. The degree 
of binding is increased in the latter cases by zinc 
ions but not by calcium or magnesium. 

MISCELLANEOUS STUDIES 

An interest in the chemotherapeutic effect of 
riboflavin antagonists has characterized Dr. 
Lane's activities for the last 5 years because some 
of these drugs retarded the growth of certain rat 
neoplasms but did not produce riboflavin de- 
ficiency in man (Annual Reports 1955, 1956). 
The Upjohn Company cooperated most gener- 
ously in these studies, and some time ago Merck 
and Company made a different analog of ribo- 
flavin— galactoflavin — available for this research. 
Galactoflavin has produced typical riboflavin de- 
ficiency in two patients with disseminated cancer, 
and it may now be possible to determine the effect 
of this deficiency state on the growth of clinical 
cancers. 

Limited experience in the treatment of cancer 
patients with either methyl-glyoxol-bis-guanylhy- 
drozone or narcotine has not elicited any favorable 
responses. 

A compound related to actidione, known as E37, 
has been studied by Dr. Rail in conjunction with 
the Hazleton laboratories. Female rats are more 



42 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



susceptible to its toxic effect than are male rats, but 
such sex differences are not found in dogs. This 
drug which Dr. Goldin finds effective against leu- 
kemia L1210 produces a peculiar hemorrhagic and 
necrotic lesion in the lungs of dogs similar to the 
lesion described by investigators at the Sloan-Ket- 
tering Institute in the lungs of patients who had 
received this agent. 

Service Functions 

Requests for consultation by members of the 
Surgery Branch, NCI, have increased from 294 in 
1957 to 437 in 1959. All of these requests are proc- 
essed through Dr. Smith's office. The greatest 
change in demand has been for urological services 
which has increased 7 times over the 1957 figure. 
It is difficult to recruit and retain a staff of capable 
surgeons because of the obvious economic advan- 
tages of private or group practice. 

The same considerations apply in an important 
degree to the generality of our senior clinical in- 
vestigators. Our pay scale is just too low in rela- 
tion to the opportunities that university medical 
schools can provide. 

Mr. Joseph Albrecht and his group of excellent 
histopathology technicians have provided this 
staff with a total of 154,000 stained histological 
preparations of which 20,000 required special 
stains. The average cost was about $1.12 per slide, 
regardless of the stain used. This is an unusually 
low figure, especially when one considers that the 
cost of training unskilled recruits is included. 

Members of the Pathologic Anatomy Branch 
performed 265 necropsies during 1959 of which 
161 were performed for NCI. They examined 
2,601 surgical specimens and accessioned 3,547 



cytodiagnosis specimens. Many of their research 
contributions are included in the foregoing mate- 
rial. 

Mr. R. J. Koegel's analytical group in the Lab- 
oratory of Biochemistry accepted 50 percent more 
specimens for microchemical analysis during 1959, 
despite a decrease of 15 percent in available man 
hours. His program on infrared spectrophoto- 
metric research has operated at a reduced level, 
but 20 percent of the samples submitted for em- 
pirical analysis are now studied by infrared tech- 
niques for purposes of structural interpretation 
and as an indication of chemical purity. 

The Animal Production Section of the Labora- 
tory Aids Branch, DRS, converted to a fee-for- 
service basis in July 1958. The staff of NCI was 
therefore required to predict the need for mice, 
rats, guinea pigs, hamsters, and rabbits for an 
entire year. The prediction proved correct with- 
in 10 percent of actual usage. This is no small 
feat, when one considers the number of persons in- 
volved, and emphasizes the ability and desire of 
the intramural research staff to accept responsi- 
bility, discharge it most capably, and cooperate 
with other organizational segments of NIH in 
solving difficult problems of mutual interest. 

This long report is liberally sprinkled with 
problems requiring the attention of many people. 
Our colleagues are creative. The information is- 
suing from the clinic and the laboratories is im- 
portant to the cancer patient; yet some of it 
requires a period of further development to assay 
its utility. We would prefer to accomplish this 
within the framework of a direct or contractual 
operation of NCI and hope that we may be en- 
couraged to do so. 



NATIONAL HEART INSTITUTE 



The Heart Institute is charged with responsi- 
bility for research aimed at improving methods 
for prevention and treatment of disorders of the 
cardiovascular system. 

Organization of intramural research in the 
Heart Institute is based on the premise that prog- 
ress toward any goal in science is best made by 
the creative efforts of individuals motivated by 
their own intellectual curiosity toward the solu- 
tion of problems that interest them. It does not 
seem appropriate to attempt to divide research 
into the categories of "applied" or "basic," since 
there would be little agreement on how such a 
classification could be made or even on how the 
groups would be defined. One suspects that the 
scientist himself would accept the proposition that 
the man working on his own problems and follow- 
ing whatever leads may arise is doing basic re- 
search, while the man working on problems de- 
vised by someone else is doing applied research. 

The problems which interest some individuals 
may have immediate practical significance; that 
which motivates others may not. Thus, within the 
Heart Institute there are men interested in im- 
proving the treatment of hypertension, in the im- 
provement of diagnostic technics and surgical pro- 
cedures for the correction of anatomical defects 
of the heart, while others are concerned with ex- 
ploring the mechanism of chemical reactions or 
the relation between the structure of a protein and 
its biologic function. 

Only through both types of activity can the 
long-term goals be achieved and the best assurance 
of maximum progress is a high level of general 
scientific productivity. The major responsibility 
of those charged with the leadership of intramural 
research is to assure maximum productivity by 
the selection of men (for the promise of their areas 
of interest as well as their capacity to contribute) 
and the provision of an environment and facilities 
most conducive to scientific accomplishment and 
interdisciplinary collaboration. 

The pages that follow reflect the scientific prog- 
ress within the individual research groups of the 



Heart Institute in the last year largely as seen by 
the leaders of those groups. 

Laboratory of Cellular Physiology and 
Metabolism 

SECTION ON CELLULAR PHYSIOLOGY 

The work of the Laboratory of Cellular Physi- 
ology and Metabolism, Section on Cellular Physi- 
ology, continues to be aimed at elucidation of 
protein structure, the relationships between this 
structure and specific function, and with the syn- 
thesis of proteins with respect both to biochemical 
mechanism and genetic control. 

During the past year the work of the Section 
dealt with these areas: (1) The development of 
methods for the study of protein structure and the 
application of these methods to ribonuclease, 
lysozyme, and several other proteins. These 
studies also interlock with investigations on the 
relationships between structure and function in 
biologically active proteins ; (2) Investigations of 
the genetic control of the biosynthesis of proteins 
of bacteriophage with emphasis on the enzyme 
lysozyme, a catalyst employed by the phage par- 
ticles for rupturing the cell wall of their host bac- 
terial cell; (3) Investigations of the secondary 
and tertiary structure of certain fibrous proteins 
and fibrous protein models; (4) Biosynthesis of 
proteins in the hen's oviduct and a detailed study 
of certain lipid substances which appear to be 
intimately involved with the biosynthetic process ; 
and (5) Studies on the metabolism of triglycerides 
by adipose tissue and liver. 

(1) The complete structure of ribonuclease has 
now been worked out in detail, through the com- 
bined efforts of Dr. Werner Hirs and his colleagues 
at the Rockefeller Institute, and Dr. Anfinsen and 
his colleagues of the Section on Cellular Physi- 
ology. Certain inconsistencies between the results 
of the two groups have been investigated in some 
detail and resolved in the past few months. These 
inconsistencies were concerned mainly with two 
of the 124 amino acid residues of ribonuclease 

43 



44 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



whose positions in the polypeptide chain, as re- 
ported by Hirs et al., required inversion on the 
basis of the NHI data. This relatively minor 
point has been examined by a series of controlled 
proteolytic digestions and quantitative analyses. 
These detailed studies were of special interest 
since they involve the portion of the polypeptide 
chain which other studies suggest is involved in 
the enzyme's active center. 

In a continuation of earlier research it has been 
shown that all four disulfide bridges in ribonu- 
clease can be cleaved by reduction with mercapto- 
ethanol and that the resulting inactive product can 
be converted to the orginal native molecule by sim- 
ple exposure to atmospheric oxygen. Earlier un- 
certainties regarding the proper matching of half- 
cystine residues have now been resolved by the 
demonstration that the pairing of such sulfhydryl 
side chains is almost certainly identical with that 
found in the native protein and by the demonstra- 
tion that regenerated protein is indistinguishable 
from native protein in immunochemical cross re- 
actions. It has also been possible to show that 
approximately one-fifth of the polypeptide chain 
of the native molecule can be removed before the 
reduction-reoxidation procedui'e without destroy- 
ing the "regeneratability" of the disulfide bonds 
in the remaining four-fifths of the protein. In 
terms of "genetic information," therefore, it seems 
possible to state that the information necessary for 
proper disulfide bridge formation is coded into 
only four-fifths of the molecule and that the rest 
of the chain must be present for other biological 
reasons. These studies are being continued with 
the aim of reducing the protein to a minimum size 
which will still permit reduction and reoxidation 
with the formation of an active regenerated sub- 
stance. It is also planned to continue the work on 
stepwise reduction and stepwise reoxidation in an 
effort to prepare active intermediates which differ 
significantly in gross structure from the native 
enzyme. Preliminary studies have already indi- 
cated that several amino acids at the ends of the 
reduced, extended chain are superfluous from the 
standpoint of function and more drastic degrada- 
tion is therefore indicated. 

The species comparisons which showed differ- 
ences in structure between sheep and beef ribonu- 
clease — as reported in last year's annual report — 
now have been extended to porcine pancreatic 
ribonuclease. Although the latter is superficially 



identical with the bovine enzyme in covalent struc- 
ture it is totally nonreactive with antiserum pre- 
pared against bovine ribonuclease (with first 
course serum, but reactive with second and third 
course serum). These immunological observa- 
tions indicate the necessity for a more detailed 
study of comparative structure since they suggest 
that there may be some drastic, but not obvious, 
difference, perhaps in the nature of the pairing of 
disulfide bridges. Highly purified preparations of 
ribonuclease have also been made from spinach 
leaves and B. subtilis, although not in sufficient 
quantities or purity for attempting structural 
analyses. The species comparisons will be contin- 
ued since they should lead to information on com- 
mon denominators of structure which would in- 
ferentially suggest the location and nature of the 
active center of ribonucleases in general. 

The variation of protein structure among vari- 
ous species is also being studied by preparing ly- 
sozymes from several bacteriophages and from 
the egg whites of a broad spectrum of birds. 
These studies, being carried out in part in collabo- 
ration with Professor Charles Sibley at Cornell, 
are at the moment mainly concerned with the de- 
velopment of simple, reproducible methods for 
the isolation of lysozyme from egg whites and 
such a method is now essentially free of difficul- 
ties. It involves adsorption of the very basic ly- 
sozyme protein onto the cation exchanger XE-64, 
followed by elution and purification on columns of 
the same resin. Comparisons of structure can 
then be made on the purified proteins by separa- 
tion of peptides produced by tryptic and chymo- 
tryptic digests on paper sheets, using chromato- 
graphic and electrophoretic methods — the so- 
called "fingerprinting" technique. Preliminary 
results already indicate that the lysozymes from 
species to species will vary considerably less in 
structure than ovalbumins from the same species, 
supporting the hypothesis that enzymes can, in 
general, suffer less change during evolution than 
proteins whose functions are more concerned with 
storage or cellular architecture. 

(2) A major effort is being made to determine 
whether or not there exists a direct correspond- 
ence between the arrangement of genetic subunits 
in specific genes and the structure of the protein 
controlled by this particular gene. Since lyso- 
zyme from bacteriophages is a relatively small 



NATIONAL HEART INSTITUTE 



45 



and easily isolated protein, and since mutant 
forms of bacteriophages should be relatively easy 
to isolate and subject to genetic mapping, this 
protein has been chosen for special study. A 
number of mutant forms of bacteriophage T2 and 
T4 have been isolated which, during growth, re- 
lease lysozymes of varying heat stability into the 
surrounding medium. It is the present plan to 
make genetic crosses of these mutants for the pur- 
pose of gene mapping and to isolate the lysozymes 
in pure form for direct comparison of structures. 
A major aspect of the work at the moment in- 
volves the study of the sequential structure of 
both bacteriophage and egg-white lysozymes in 
order to provide baselines for future studies on 
the relationships between structure and function. 
It is also planned to investigate whether or not 
nonlethal mutations will, as might be predicted, 
only occur in those areas of the lysozyme molecule 
that are not essential for activity. 

(3) The gross molecular structure of myosin, 
the protein unit of the muscle contractile mech- 
anism, has been under active study by Drs. Har- 
rington and Mihalyi. It was observed that the 
molecular weight of the myosin particle was de- 
creased from 619,000 to 206,000 by concentrated 
guanidine solutions which tend to rupture hy- 
drogen bonds and break protein polymers into 
smaller units. The conclusions reached from 
ultra-centrifugation studies were supported by 
sedimentation, diffusion and viscosity measure- 
ments. These results, when considered together 
with observed length and width and with the 
optical rotatory and X-ray diffraction properties 
of the myosin molecule, led to the conclusion that 
myosin is made up of three identical polypeptide 
chains, each wound into an a-helix and with the 
three strands twisted together to form a rope-like 
structure. Studies of the primary sequence of 
the myosin unit chain are now in progress. These 
should reveal whether all three are oriented in the 
same direction. The details of primary structure 
may then be used to explain the secondary and 
tertiary coiling and folding. Efforts are also be- 
ing directed at determining the mechanism by 
which myosin subunits polymerize to form the ag- 
gregates characteristic of myofibrils. A second 
group of fibrous protein or protein-like molecules 
that have been thoroughly investigated are the col- 
lagens and collagen-like proline-glycine copoly- 



mers. Various proteolytic enzymes have been em- 
ployed as specific probes of the second structure 
(that is, the internal coiling) of these long poly- 
peptide chains and it appears that here, as in the 
case of myosin, these long molecules are made up 
of alternating amorphous and semicrystalline re- 
gions with differential sensitivity to proteolysis. 
The kinetics of proteolytic digestion suggests that 
neighboring charged groups strongly influence the 
susceptibility of sensitive bonds to hydrolysis. 

(4) In the biosynthesis of proteins in the hen's 
oviduct, certain lipid components appear to be as- 
sociated with an extremely active pool of amino 
acids. Dr. Hendler has separated these on 
alumina-silica columns in quantities for direct 
chemical study. When oviduct tissue or the 
bacterium E. coli is incubated with radioactive 
amino acids, the first metabolic pool to become 
labeled is a class of organic soluble substances 
which carry amino acids and peptide-like com- 
pounds. Whether or not the bond between the 
amino acids and peptides and the lipid moieties 
is covalent has not been established. The com- 
bined information on these interesting compounds 
suggests that they may be involved in the biosyn- 
thetic processes taking place in the so-called endo- 
plasmic reticulum, which, it has been suggested, 
may be associated with protein synthesis. A 
study on the dissociation of this endoplasmic 
reticulum into its lipid components and ribosomal 
granules and the subsequent separation of these 
various components on ion exchange columns has 
been undertaken in collaboration with Drs. Peter- 
son and Kuff of the National Cancer Institute. It 
is believed that information for making particular 
proteins is contained in the configuration of the 
nucleic acids of the ribosomal granules. On this 
basis one should expect that these granules will 
be heterogeneous and that it may be possible to 
fractionate them into classes, each responsible for 
a particular protein or group of proteins. 

(5) Work on triglyceride metabolism and on 
the nature of the heparin-induced lipoprotein li- 
pase — under investigation in this laboratory for a 
number of years — has been continued by Dr. Korn. 
Lipoprotein lipase has been subjected to further 
purification with the purpose of determining 
whether heparin is an integral part of the enzyme. 

Another group of long-chain polysaccharides 
associated with the yeast cell wall has also been 



46 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



investigated. These experiments, carried out by 
Dr. Korn in collaboration with Dr. D. H. North- 
cote at Cambridge University, have led to the 
isolation of fractions of yeast cell walls much 
more highly purified and better characterized than 
has previously been obtained. The studies on cell 
wall chemistry serve as models for the study and 
understanding of other conjugated proteins. The 
techniques for handling large conjugated proteins 
are relatively similar, whether the conjugated ma- 
terial is lipid or carbohydrate, and these studies 
should serve, therefore, as excellent background 
for the projected investigations of lipoproteins. 

Dr. Rodbell has continued his investigation of 
the metabolic processes involved in the removal 
of chylomicrons of plasma. Rat epididymal adi- 
pose tissue does not distinguish between rat chylo- 
microns and synthetic fat emulsions with respect 
to uptake and metabolism, suggesting that chy- 
lomicron proteins are not essential for fat uptake 
or metabolism. Inhibition of lipoprotein lipase 
did not substantially reduce fat uptake, suggest- 
ing that this enzyme is perhaps necessary for chy- 
lomicron metabolism but not for transport into 
cells. C 14 -labeled triglycerides were taken up 
from blood by the parenchymal cells of rat liver 
and these triglycerides were found to be associated 
with the microsomes and nuclei of the liver cells. 
The triglycerides are then converted to phospho- 
lipids and triglycerides characteristic of normal 
liver fats. These studies, in general, have impli- 
cated the microsomes in the absorption of exogen- 
ous fat by liver cells and it would appear that 
the endoplasmic reticulum may serve both as a 
channel for the entry of exogenous triglycerides 
as well as the site for metabolism and transforma- 
tion. 

SECTION ON METABOLISM 

Studies on the basic physiology of fat absorption 
and fat transport 

(a) Considerable progress has been made in 
studies of the metabolism of adipose tissue and 
the nature of its responses to hormonal factors. 
Last year it was reported that epinephrine added 
in vitro would stimulate the release of free fatty 
acids from adipose tissue. It has now been shown 
that glucagon and ACTH added in vitro also 
stimulate release of free fatty acids. Further 



studies revealed that all three of these hormones 
lead to an increase in the levels of active phos- 
phorylase in adipose tissue and stimulate the up- 
take of glucose. It is of interest that this activity 
in the adipose tissue is quite analogous to the ac- 
tivities of these hormones on other peripheral 
tissues. Epinephrine and glucagon increase phos- 
phorylase activity in the liver (but not in the 
adrenal) and ACTH increases phosphorylase 
activity in the adrenal (but not in the liver) . Pre- 
liminary results suggested that these hormones 
might effect the observed increase in rate of re- 
lease of fatty acids by inhibiting the synthesis of 
triglycerides. For this reason studies on the 
mechanism of triglyceride synthesis in adipose 
tissue were initiated. A cell-free system which 
will incorporate fatty acids into triglyceride has 
been derived from rat epididymal fat pads. This 
system carries out the first reported triglyceride 
synthesis in adipose tissue homogenates. The sys- 
tem requires a-glycerophosphate as a precursor 
and glycerol will not substitute for this require- 
ment. ATP and Coenzyme A are required, pre- 
sumably for the activation of the free fatty acids. 
Diglycerides of very high specific radioactivity 
have been isolated and are probably intermedi- 
ates. Unlike the system in liver the adipose tissue 
homogenate does not accumulate phosphatidic 
acid but the requirement for a-glycerophosphate 
suggests that this is nevertheless an intermediate 
in the synthetic pathway. These studies are being 
pursued in the hope that with a better under- 
standing of adipose tissue metabolism it may be 
possible to demonstrate the site at which the sev- 
eral hormones discussed above interact with the 
enzymatic mechanisms controlling fat deposition 
and release. 

Heparin is known to lead to a marked increase 
in the levels of lipoprotein lipase in the serum. 
Studies completed this year show that addition of 
heparin to adipose tissue in vitro leads to a strik- 
ing outpouring of lipoprotein lipase from the 
tissue into the medium. Also of interest is the 
finding that the levels of lipoprotein lipase in the 
adipose tissue of fasting rats is considerably lower 
than the level found in the tissues of carbohy- 
drate-fed rats. Thus, the levels of lipoprotein 
lipase, rather than paralleling the rate of release 
of fatty acids, vary inversely with the rate of 
release of fatty acids. These results suggest that 



NATIONAL HEART INSTITUTE 



47 



the role of lipoprotein lipase may be in the uptake 
of fat rather than in its release. 

(b) A study has been made of the fatty-acid 
composition of the chylomicron fat in patients fed 
large meals of different types of fat. It was found 
that the pattern of fatty acids in the chylomicron 
resembles very closely the pattern of the dietary 
fat used. These results are clearcut and disagree 
with results reported by Dole of the Rockefeller 
Institute, who claimed that there were large dif- 
ferences between the composition of fats fed and 
the fat in the chylomicrons during absorption. 
The disagreement may stem from the failure of 
the latter investigator to completely remove low 
density lipoproteins from chylomicrons prior to 
analysis. 

Administration of carbohydrate by mouth or 
intravenously reduces considerably the rate of ab- 
sorption of fat, as shown by studies carried out in 
rats with cannulated thoracic ducts. 

(c) Kinetic studies on the utilization of in- 
jected C 14 -fatty acids have been continued and 
analysis of these results shows that at least 50 
percent of the fat utilized during fasting is trans- 
ported through the serum as free fatty acid. In- 
jection of epinephrine raises the net turnover of 
free fatty acids. During exercise there is a 
marked increase in net fatty acid utilization but 
the fraction accounted for by transport through 
the FFA fraction falls considerably. 

Studies of dietary and hormonal factors deter- 
mining serum lipoprotein levels 

(a) Adrenal Control or Lipoprotein Levels. 
It has been shown that injection of epinephrine in 
oil not only elevates the plasma levels of free fatty 
acids (FFA) but also leads to an elevation of lipo- 
protein levels. The FFA response occurs early 
and is transient ; the rise in lipoproteins does not 
occur until 12 to 24 hours after epinephrine injec- 
tion. Studies completed this year showed that 
adrenalectomy or hypophysectomy abolished both 
the FFA and the lipoprotein responses to epine- 
phrine. Pretreatment of the operated animals 
with cortisone or with ACTH, respectively, re- 
stored their ability to respond to epinephrine with 
both a rise in FFA and in lipoproteins. Admini- 
stration of cortisone to normal dogs exaggerated 
the lipoprotein response to epinephrine injection. 



When the animals received extra cortisone as 
much as an 80 percent rise in serum cholesterol was 
obtained with three daily injections of epine- 
phrine. 

These results suggest a physiologic basis for the 
hypercholesterolemia of stress. It is well known 
that animals and patients under stress demon- 
strate hyperactivity of both the adrenal medulla 
and the adrenal cortex. This pattern of hormone 
production would appear to be adequate to ex- 
plain elevations of both FFA and cholesterol 
(lipoproteins). Studies are currently in progress 
to evaluate the response of patients to exogenous 
epinephrine and cortisone. 

(b) Studies of the effects of dietary fat on 
cholesterol excretion have been completed. Of 
major interest was the observation that a surpris- 
ingly large fraction of the cholesterol excreted 
in feces in man appears there in the form of cho- 
lesterol itself (35 to 80%). This is in contrast 
to the pattern in rats and other laboratory ani- 
mals in which practically all of the cholesterol 
excreted appears in the feces in the form of bile 
acids. A study of eight patients fails to reveal 
any consistent effect of unsaturated fats on the 
rate of excretion of intravenously administered 
C 14 -cholesterol in the feces. The mechanism by 
which dietary fats modify cholesterol levels has 
not been established. The effect may be on a re- 
distribution of cholesterol within the body but 
this has not been established in man. 

Parallel with the studies of cholesterol excre- 
tion, bile acid turnover studies have been done 
under various dietary conditions and in various 
clinical conditions. These studies were carried out 
in collaboration with Dr. Sven Lindstedt from 
Sweden. Results of the study are not yet com- 
plete. These collaborative studies are continuing 
in order to determine whether there are systematic 
differences in bile acid turnover in various forms 
of hypercholesterolemia. 

(c) Studies of the production of lipoproteins 
by rat liver slices in vitro were continued and de- 
finitive identification of alpha-1-lipoprotein was 
obtained. This was done by preparing lipopro- 
teins in vitro from a complete mixture of C 1 *- 
amino acids, purifying them, digesting with 
trypsin and chymotrypsin, and chromatographing 
the mixture of peptides in two dimensions. It 
was found that all of the peptides derived from 



48 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the alpha-1-lipoprotein coincided with peptides 
derived from alpha- 1 -lipoprotein prepared from 
normal rat serum. The identification of the beta- 
lipoproteins with serum beta-lipoproteins was in- 
conclusive. 

It was shown that the rate of cholesterol syn- 
thesis is not apparently a rate-limiting reaction in 
lipoprotein synthesis. Liver slices taken from 
cholesterol-fed rats (in which the rate of choles- 
terol synthesis is markedly suppressed) incor- 
porated labeled amino acids into the protein 
moiety of lipoproteins at a normal rate. Con- 
versely, accelerating the rate of cholesterol synthe- 
sis by injection of Triton did not increase the rate 
of synthesis of lipoprotein protein. 

(d) In collaboration with investigators at the 
University of Maryland a study of modified milk 
fat was carried out. Dr. Shaw and his coworkers 
in the dairy department at Maryland University 
were able to alter the iodine number of milk fat 
by appropriate changes in feed. However, the 
changes were relatively small (increase in iodine 
number from 30 to 48) and no significant differ- 
ence in the effects of fat of these two types was 
demonstrable in patients. Thus, it appears that 
unless a more radical change can be effected this 
approach to the problem of dietary fat will not 
be suitable. 

(e) The technique previously described for in- 
corporating cholesterol into lipoproteins has 
proved valuable for the incorporation of other 
nonpolar molecules. In particular, the technique 
serves to incorporate carcinogenic hydrocarbons 
so that these can be administered intravenously 
in known quantities and their metabolism studied. 
Studies of these hydrocarbons have been hampered 
because of their insolubility and the resultant un- 
certainty in evaluating absorption and distribu- 
tion. 

Studies on the metabolism of cholesterol and 
therapeutic agents useful in lowering serum 
cholesterol levels 

(a) A new inhibitor of cholesterol biosynthesis 
produced by the Wm. S. Merrell Co. (MER-29) 
has been studied in animals and in man. This 
compound — 1 - [p - (/? - diethylaminoethoxy) - 
phenyl]-l- (p-tolyl) -2- (p-chlorophenyl) ethanol — 
was shown by Dr. Blohm to suppress markedly 
the incorporation of radioactive acetate into 
cholesterol and to lower the serum and tissue levels 



of cholesterol in rats. Studies in this laboratory 
with the collaboration of Dr. Erich Mosettig and 
Mr. Thompson of the Arthritis Institute have now 
established the probable site of action of the drug. 
It has been shown that 24-dehydrocholesterol ac- 
cumulates in the liver of rats fed MER-29. It 
may account for as much as one-half of the total 
sterol in these livers. 24-dehydrocholesterol 
(desmosterol) has previously been shown to be a 
precursor of cholesterol in the rat. It differs from 
cholesterol only in having an additional double 
bond at the 24, 25 position in the side chain. Pre- 
sumably it is converted to cholesterol by a simple 
reduction step. It will be of interest to explore 
the mechanism by which this new drug blocks this 
last step in cholesterol synthesis. 

Clinical studies confirmed the work of others 
in that there was some lowering of serum 
cholesterol levels, although this was not marked. 
Some patients were studied on a diet free of 
cholesterol but this did not appear to magnify the 
response of the drug. It was possible to show 
that 24-dehydrocholesterol appears in the serum of 
treated patients in significant amounts. Because 
this sterol gives a lower color yield in the Lieber- 
man-Burchard reaction the apparent drop in 
serum cholesterol obtained using the usual meth- 
ods is misleading. While there is a slight decrease 
in total sterol it is smaller than would appear from 
the usual analyses. It will be important to evalu- 
ate the atherogenic potential as well as other 
metabolic effects of 24-dehydrocholesterol before 
extending clinical trials. 

(b) A kinetic study of the distribution of 
C 14 -cholesterol among the various tissues of the 
animal organism (rat and rabbit) has shown that 
every tissue, including brain tissue, takes up radio- 
active cholesterol from the serum. By extending 
the studies over a long time period it was shown 
for the first time that the specific radioactivity of 
the slowly metabolized cholesterol pools (brain, 
muscle, kidney) contained cholesterol of a higher 
specific radioactivity than that in the serum in the 
latter stages of the experiments. A simple math- 
ematical model satisfactorily accounts for the ob- 
served results on the basis of isotopic exchange. 

Studies on hypoalbuminemia and the mecha- 
nisms responsible for it 

(a) A new clinical syndrome, exudative en- 
teropathy, or protein-losing gastro-enteropathy, 



NATIONAL HEART INSTITUTE 



49 



has been described. This is a condition charac- 
terized by loss of plasma proteins into the intestine 
with a resultant lowering primarily of the level 
of serum albumin, but also that of other serum 
proteins as well. The patients in this category 
have previously been described as having "idio- 
pathic hypercatabolic hypoproteinemia." By the 
use of a non-metabolizable polymer of molecular 
size comparable to that of albumin it has been 
shown that these patients lose into the G.I. tract 
much larger amounts of injected macromolecules 
than do normals. The fate of the polymer, poly- 
vinylpyrrolidone, which is of molecular size com- 
parable to that of serum albumin, probably re- 
flects quite well the fate of circulating albumin 
molecules. 

The diagnostic test using I 131 labeled PVP is 
technically simple. Many medical centers have 
received samples prepared at NIH and a large 
number of cases have already been uncovered. A 
commercial firm is planning to produce labeled 
polymer for routine clinical use. 

(b) Biopsies of intestinal mucosa were obtained 
in 6 cases of protein-losing gastroenteropathy. 
In 5 of these a common lesion consisting of mark- 
edly dilated lymphatics within the villi was de- 
monstrable. This, combined with the fact that 
many cases have had chylous effusions, suggests 
that there may be a common etiology somehow 
associated with the lymphatic system. 

(c) Dr. Gordon carried out similar studies in 
patients with Asiatic cholera in Bangkok. There 
was not evidence of excessive loss of protein into 
the intestinal tract. This negative finding is not 
consistent with the generally accepted concept that 
there is serious desquamation of the intestinal 
mucosa in cholera. 

Studies on the mechanisms of protein synthesis 
and degradation 

Conclusive evidence of the incorporation of 
amino acid analogues into crystalline proteins was 
obtained and published. A comprehensive review 
of "The Specificity of Protein Biosynthesis" was 
prepared and published in Advances in Protein 
Chemistry. Preliminary studies that have re- 
vealed the presence in mammalian tissue of pep- 
tides apparently conjugated to nucleotides were 
completed and published. This phase of the lab- 



oratory program has now been, temporarily at 
least, discontinued. 

Basic studies on the structure of proteins and 
the nature of the clotting process 

Investigations of the fundamental mechanism 
of fibrin formation were continued with particu- 
lar reference to the proposed role of tyrosine 
residues. By the application of highly sophisti- 
cated spectrophotometric methods it was shown 
that the tyrosine residues not titrated in fibrinogen 
are probably not involved in hydrogen bonds but 
rather masked by some sort of hydrophobic bond- 
ing. These studies are being continued, using 
careful kinetic analysis of pH changes in order 
to clarify the mechanisms of the fibrinogen-fibrin 
transformation. 

Studies on the disturbed metabolism of lipids in 
nephrosis and on the immunochemical mecha- 
nisms involved 

(a) A comprehensive study of the serum lipid 
pattern in patients with nephrosis has forced a 
revision of the usual concept that only the very 
low density lipoproteins are elevated. Many 
patients were found to have the most marked ele- 
vation in the /^-lipoprotein fraction. It was ob- 
served that during improvement due to steroid 
therapy the lipoprotein pattern undergoes shifts 
toward the higher density /^-lipoproteins. These 
findings refute the theory of Gitlin that the defect 
in nephrosis is a deficiency in the conversion of 
very low density lipoproteins to higher density 
lipoproteins. 

(b) It has been repeatedly shown that infusion 
of albumin lowers the lipid levels in nephrosis. 
Surprisingly it now appears from studies on 
nephrotic rats done in this laboratory that in- 
fusion of inert macromolecules such as dextran 
and polyvinylpyrrolidone also causes a decrease in 
lipid levels. 

(c) Intravenous infusions of glucose generally 
cause some decrease in serum cholesterol level 
and little change in serum triglyceride levels. In 
three cases of nephrosis, however, glucose infusion 
caused a marked rise in triglyceride levels and in 
low density lipoprotein levels. The significance 
of these results is not yet determined but will be 
investigated further. 



50 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Development of techniques for radioassay in the 
liquid scintillation spectrometer 

The new approach described in last year's re- 
port has now been in use for over a year and has 
proved to be a very valuable adjunct in radio- 
assay. It has been shown that the method has a 
wide range of applicability. It has been effective- 
ly used for assay of tritium, C 14 , Ca 45 , and it has 
been shown in pilot studies that it is applicable 
for counting P 32 and I 131 . The method has re- 
ceived wide acceptance, particularly for the assay 
of weak beta emitters. Studies are being con- 
tinued to determine whether the method can be 
used for pure gamma emitters and weak X-ray 
emitters. 

SECTION ON ENZYMES 

The activities of the Section on Enzymes have 
been directed toward elucidation of the following 
diverse fundamental biochemical processes: (1) 
the metabolism of heterocyclic compounds, (2) 
the metabolism of three carbon compounds, (3) 
cellular differentiation and protein synthesis, (4) 
anaerobic oxidative phosphorylation and electron 
transport, (5) nucleotide decomposition, (6) the 
metabolism of onium compounds, (7) the metab- 
olism of isoprene derivatives, and (8) the metab- 
olism of amino acids. 

The Metabolism of Heterocyclic Compounds 

(a) Riboflavin Degradation (Drs. E. R. Stadt- 
man, P. Z. Smyrniotis, and L. Tsai). Previous 
studies in this laboratory have shown that the 
oxidative dissimilation of riboflavin to ammonia 
and C0 2 by an aerobic bacterium involves the 
intermediary formation of l-ribityl-2,3-diketo-l, 
2,3,4-tetrahydro-6,7-dimethylquinoxaline (com- 
pound I) and 3,4-dimethyl-6-carboxy-a-pyrone 
(compound III). Evidence has now been ob- 
tained showing that 3,4-dimethyl-2,3-quinoxaline- 
diol (compound II) and oxamide are intermedi- 
ates in the conversion of compound I to compound 
III. The conversion of riboflavin to compound I 
involves a cleavage of the pyrimidine ring (ring 
C) with a stoichiometric formation of urea and 
C0 2 . This transformation is of special interest 
since from the overall chemical point of view it 
can be represented as a simple hydrolytic process ; 
however, it occurs only in the presence of molecu- 



lar oxygen. The conversion of compound I to 
compound II involves a cleavage of the N-ribityl 
linkage. This cleavage is also of unique interest 
since it too requires molecular oxygen. Although 
the exact fate of the ribityl moiety is still un- 
known, the oxygen requirement is not restricted 
to oxidative degradation of the side chain since 
oxygen is required also for the cleavage of the 
N-hydroxyethyl, N-methyl, and N-acetaldehyde 
analogues. The further degradation of com- 
pound II to a mixture of oxamide and the a- 
pyrone derivative is obviously a complicated proc- 
ess. This conversion is inhibited by arsenite, 
iodoacetate and hydroxylamine and is activated 
by various oxidizable substrates such as ethanol, 
lactate, pyruvate and glucose. Further studies 
on the mechanism of the individual reactions in 
these various transformations are in progress. 

(b) Phenazine-1-Carboxylic Acid Biosynthesis 
(Dr. M. Levitch). The bacterium Pseudomonas 
aureofaciens Kluyver offers a unique opportunity 
to investigate the biosynthesis of the heterocyclic 
phenazine ring system since this organism pro- 
duces unusually large quantities (1.0 gm/liter) 
of phenazine-1-carboxylic acid during growth. 
Further insight into the mechanism of this bio- 
synthetic process has been sought by measuring 
the incorporation of isotope carbon into phena- 
zine-1-carboxylic acid when bacterium is grown 
in a medium supplemented with various (Un- 
labeled compounds. Of numerous compounds 
tested, the most effective precursors are acetate, 
bicarbonate, alanine, serine and methionine. 
Methods for the stepwise degradation of the phen- 
azine derivative to permit a determination of the 
distribution of labeled carbon from the various 
precursor compounds are now being investigated. 
It is hoped that the results of these studies will 
suggest 'an intelligent approach to the problem of 
phenazine biosynthesis at the enzyme level. 

(c) Alkaloid Biosynthesis (Drs. E. Kravitz and 
P. R. Vagelos). Studies of the biosynthesis of 
opium alkaloids by tissue preparations of the 
poppy plant, Palaver somniferum, have been un- 
dertaken as an additional effort to obtain basic 
information on the biochemistry of heterocyclic 
compounds. The poppy plants were supplied by 
the USDA Plant Industry Station in Beltsville, 
Md. Progress to date has been restricted to the 



NATIONAL HEART INSTITUTE 



51 



development of optimal experimental conditions 
for the in vitro synthesis of alkaloids by tissue 
slices and the development of procedures for the 
isolation and separation of the various alkaloids 
produced. Alkaloid synthesis has been followed 
by measuring the incorporation of isotopic carbon 
into the alkaloid fraction after incubating the 
plant preparations with methyl-C 14 -methionine or 
/?-C 14 serine, which were introduced by the vacuum 
infiltration technique. It has been found that with 
4 to 5 week old plants the isotope is incorporated 
predominantly into narcotine and papaverine, 
whereas in older plants labeled morphine and co- 
deine were also produced (identification of the 
various alkaloids is still tentative). Studies with 
tissue slices derived from various parts of the 
plant have revealed that the roots are by far the 
most active sites of alkaloid synthesis. In future 
studies efforts will be made to develop cell-free 
preparations of roots that are capable of catalyz- 
ing alkaloid synthesis. 

An ion exchange procedure using Dowex-1-OIP 
and Dowex-50-H + has been devised for the separa- 
tion of the major opium alkaloids. 

In addition to the above investigation, studies 
have been initiated to investigate the biosynthesis 
of the ergot alkaloids by the fungus Claviceps 
purpura. 

The Metabolism of Three-carbon Compounds 

(a) Propionic Acid Oxidation (Drs. P. R. 
Vagelos and W. Sly). In previous studies, Dr. 
Vagelos has shown that cell-free enzyme prepara- 
tions of the bacterium, Clostridium kluyveri, cata- 
lyze the oxidation of propionate by a pathway in- 
volving the intermediary sequential formation of 
propionyl CoA, acrylyl CoA, /3-hydroxypropionyl 
CoA, malonylsemialdehyde CoA, and malonyl 
CoA. Further studies of this metabolism have led 
to the discovery of a curious exchange reaction be- 
tween the carboxyl group of malonyl CoA and 
added C 14 2 . This exchange is absolutely de- 
pendent upon the presence of catalytic amounts 
of an acyl CoA derivative of a saturated fatty 
acid having 4 to 16 carbon atoms. In view of 
the fact that malonyl CoA and C0 2 have been 
shown recently to be involved in the biosynthesis 
of fatty acids, it appears probable that the ob- 
served exchange reaction represents one step in 
fatty acid synthesis. The exact mechanism of 

556044—60 5 



this reaction is therefore of immediate interest 
and is under further investigation. 

The enzyme catalyzing the TPN-coupled oxida- 
tion of malonyl-semialdehyde CoA to malonyl 
CoA has been partially purified and is under fur- 
ther study. 

Incidental to these investigations has been the 
development of a good general method for the 
chemical synthesis of /3-ketothiolesters. 

(b) The Role oe Biotin and Vitamin Bi 2 -Co- 
enzyme in Propionate Metabolism (Dr. E. R. 
Stadtman in collaboration with Mr. P. Overath 
and Prof. F. Lynen in the Max Planck Institute 
fur Zellchemie, Munchen, Germany). Previous 
studies by Flavin et al., with animal enzymes, and 
studies by Whitely, Carson, Wood and Delwiche, 
with enzymes derived from propionic acid fer- 
menting bacteria, have established an intermedi- 
ary role for succinyl CoA and methylmalonyl 
CoA in the metabolism of propionic acid. A con- 
sideration of the fact that propionic acid forma- 
tion represents the major metabolic process 
catalyzed by bacteria belonging to the genus 
Propionibacteria and that these organisms pos- 
sess unusually high concentrations of vitamin B i2 
coenzyme and biotin, have prompted an investi- 
gation to determine if these vitamins are involved 
in propionic acid metabolism. Propionic acid 
metabolism in cell-free extracts of Propionibac- 
terium s'hermanii was measured by the overall in- 
corporation of l-C 14 -proprionate into succinate. 
After treatment with protamine and charcoal and 
then dialysis, cell-free extracts lose their ability to 
catalyze the incorporation of labelled propionate 
into succinate. This ability is restored by the ad- 
dition of catalytic levels of acetyl CoA and a 
light-labile factor present in boiled extracts. 
The latter factor is completely replaced with low 
concentrations (10 _8 M) of pure dimethylben- 
zyimidazole-Bi 2 -coenzyme (supplied by H. A. 
Barker). A role of biotin in the propionate ex- 
change system is indicated by the fact that the 
reactivated enzyme is completely inhibited by 
avidin but not by avidin which has been pretreated 
with biotin. In light of the recent report that the 
succinyl- CoA isomerase activity of rat liver is 
lowered in Bi 2 deficiency, the above findings form 
the basis of a working hypothesis that the pro- 
pionate-succinate exchange is the net result of two 



52 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



vitamin coenzyme-linked reactions: (1) the B 12 - 
coenzyme dependent isomerization of succinyl CoA 
to form methylmalonyl CoA, and (2) the reaction 
of methylmalonyl CoA with biotin-enzyme to 
form a biotin-enzyme- C0 2 complex and propionyl 
CoA. The reversible exchange of propionyl CoA 
with free labeled propionate and reversibility of 
the other postulated reactions could account for 
the observed results. This hypothesis is under 
investigation. 

(c) Propionic Acid Fermentation by Clos- 
tridium Propionicum (Dr. H. Goldfine). In 
continuing studies on the anaerobic fermentation 
of three carbon compounds it was found that cell- 
free extracts of 0. propionicum convert pyruvate, 
lactate and serine predominantly to acetate and 
C0 2 , whereas a-alanine and /^-alanine are con- 
verted mainly to propionate. Evidence was ob- 
tained supporting the conclusion that propionate 
formation from ^-alanine proceeds by the follow- 
ing pathway : /? - alanine— >/? - hydroxypropionate 
^/3-hydroxypropionyl CoA— >acrylyl CoA— >pro- 
pionyl CoA— ^propionate. The first step, i.e., the 
conversion of /3-alanine to /?-hydroxypropionate 
involves the release of ammonia and is obligately 
dependent upon the presence of catalytic amounts 
of pyruvate and a-ketoglutarate. The latter ob- 
servation and the demonstration that /3-alanine 
serves as an amino group donor to form a-alanine 
and glutamate from the corresponding a-keto- 
acids together with the further discovery that 
the DPN-linked oxidative deamination of a-ala- 
nine occurs only in the presence of a-ketogluta- 
rate, supports the conclusion that the formation 
of /?-hydroxypropionate from /3-alanine occurs by 
a transamination of the amino group of /^-alanine 
to pyruvate, thence to a-ketoglutarate, and then 
the release of the amino group as free ammonia 
by the action of glutamic dehydrogenase. As yet 
no evidence has been obtained for the formation 
of malonyl semialdehyde as the expected inter- 
mediary in /3-alanine transamination. 

The Biochemistry of Cellular Differentiation and 
Protein Synthesis. 

(a) Amino Acid and Protein Metabolism in the 
Slime Mold (Dr. B. K. Wright, Mr. G. McNeil 
and Miss Minnie Anderson) . Studies on the turn- 
over of amino acids and protein during cellular 



differentiation of the slime mold D. discoideum 
have been continued. It has been found that the 
differentiation process is associated with a net de- 
crease in protein content, but that active protein 
synthesis, as measured by the incorporation of 
S 35 methionine into the protein fraction, occurs 
throughout all stages of development. At precul- 
mination the methionine in protein is replaced by 
the endogenous pool S 35 -methionine at a rate of 
about 7 percent per hour. A unique feature of 
this metabolic system is the discovery that the 
size of the "free" endogenous methionine pool is 
not influenced by changes in the exogenous me- 
thionine concentration. Although fixed in size at 
any given stage of development, the endogenous 
methionine pool can nevertheless undergo ex- 
change with exogenous S 35 -methionine, and the 
extent of this exchange (i.e., the specific isotope 
content of the pool methionine at equilibrium) is 
a linear function of the exogenous S 35 -methionine 
concentration. This curious phenomenon remains 
as yet unexplained. The results suggest the possi- 
ble existence of a heterogeneous endogenous me- 
thionine pool, in which the exchangeability of var- 
ious parts is differentially influenced by the ex- 
ternal methionine concentration. 

Following momentary exposure of the organism 
to S 35 methionine, the separation of cellular pro- 
teins into various arbitrary classes by means of 
solubility in ethanol and by DEAE column 
chromatography has revealed marked differences 
in the rates of isotope incorporation into the var- 
ious protein classes. From such studies evidence 
has accumulated which indicates that methionine 
molecules in various parts of the amino acid pool 
are "fixed" with respect to the proteins into which 
they are incorporated. It appears that, on the 
average, pool methionine molecules which ex- 
change readily with exogenous S 3S -methionine are 
most readily incorporated into certain protein 
fractions which attain a relatively high specific 
radioactivity, whereas pool methionine molecules 
exchanging poorly with exogenous S 35 -methionine 
are preferentially incorporated into protein frac- 
tions attaining a relatively low specific radioac- 
tivity. It is evident from the results obtained that 
the slime mold is particularly well suited for fur- 
ther studies on the biochemistry of protein metab- 
olism. 



NATIONAL HEART INSTITUTE 



53 



(b) The Chemotactic Hormone, Acrasin (Dr. 
B. K. Wright and Mr. G. Liddel, in collaboration 
with Dr. E. Heftmann of NIAMD). Acrasin is 
the chemotactic hormone involved in initiation of 
aggregation at the onset of differentiation. A 
sterol with acrasin activity has been isolated from 
D. discoideum as a pure crystalline compound and 
has been identified as A 22 -stigmasten-3B-ol. As 
judged by the lack of hormone activity in other 
fractions during purification and by the fact that 
the recovered acrasin accounts for most of the 
hormone activity of the crude cellular extract, it 
is concluded that this sterol is the major active 
compound present after acid hydrolysis. Since it 
is not as active as crude acrasin, attempts to isolate 
a conjugated form of this sterol are in progress. 

Anaerobic Oxidative Phosphorylation and Elec- 
tron Transport (Drs. E. B. Brown and E. R. 
Stadtman). 

The reduction of crotonyl CoA to butyryl CoA 
by reduced pyridine nucleotide is associated with 
a standard free energy change of — 14,000 calories 
and it has been postulated that this oxido-reduc- 
tion system may be coupled with phosphorylation. 
In preliminary reports from another laboratory 
evidence has been presented to support the con- 
clusion that ATP is produced during the reduc- 
tion of crotonyl CoA to butyryl CoA by extracts 
of Clostridium hluyveri. Results of the present 
studies on this enzyme system suggest that the ob- 
served phosphorylation may not be associated with 
the reduction of crotonyl CoA per se but that it 
is derived indirectly by a dismutation of crotonyl 
CoA to butyryl CoA and acetyl CoA, followed by 
the formation of ATP from the latter compound 
via acetyl phosphate. 

Nucleotide Decomposition (Drs. E. B. Brown and 
E .R. Stadtman) . 

Four separate ferrous iron-dependent nucleo- 
tidases were identified and partially purified from 
cell-free extracts of G. propionicum. Two of these 
enzymes are mononucleotidases sharing a remark- 
able degree of resistance to heat but differing in 
their sensitivity to versene inhibition; the other 
pair of enzymes are heat sensitive dinucleotidases 
separable on the basis of versene sensitivity. Suc- 
cessive action of the di- and mono-nucleotidases 
catalyzes the irreversible decomposition of diphos- 
phopyridine nucleotide to adenosine, nicotinamide 



mononucleotide and two equivalents of orthophos- 
phate. 

The Metabolism of Onium Compounds 

(a) The Anaerobic Fermentation of Choline 
(Drs. H. Hayward and T. C. Stadtman). An or- 
ganism capable of deriving its carbon, nitrogen 
and energy for growth from the anaerobic dis- 
similation of choline was previously isolated from 
the soil and was shown to catalyze the conversion 
of choline to one mole of trimethylamine and one- 
half mole each of acetate and ethanol. This or- 
ganism has now been identified as a new species 
belonging to the genus Vibrio and has been given 
the name Vibrio cholinicus. Studies with cell- 
free extracts of the organism have shown that 
choline degradation involves the intermediary 
formation of acetaldehyde which then undergoes 
a dismutation to form ethanol and acetate. By 
means of sedimentation in an ultracentrifuge, 
crude sonic extracts have been separated into a 
particulate fraction and a soluble fraction, both 
of which are needed to catalyze the decomposition 
of choline. The enzymes catalyzing the dismuta- 
tion of acetaldehyde are present in the soluble 
fraction. This dismutation is catalyzed by the 
joint action of a TPN-specific ethanol dehydro- 
genase and an acetaldehyde dehydrogenase. Al- 
though the detailed mechanism of acetaldehyde 
oxidation has not been elaborated, it is significant 
that no dismutation occurs in the absence of TPN, 
ADP or a sulfhydryl compound; moreover, the 
reaction is markedly stimulated by the addition of 
ferrous iron or other divalent cations and by co- 
enzyme A. 

It has been further established that the dis- 
similation of choline by crude extracts is asso- 
ciated with the esterification of orthophospate to 
form ATP. The possibility that this phosphory- 
lation is associated with electron transport is sug- 
gested by the fact that phosphorylation is inhib- 
ited by 2,4-dinitrophenol in concentrations known 
to uncouple oxidative phosphorylation. Prelimi- 
nary, as yet inconclusive, evidence has been ob- 
tained that betainaldehyde is an intermediary in 
choline degradation. 

The discovery that cell-free extracts of the 
Vibrio organism contain large amounts of a cyto- 
chrome pigment spectrally similar to animal cyto- 
chrome c was previously reported. A functional 



54 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



role of this cytochrome in choline metabolism is 
suggested by the observation that cell-free extracts 
catalyze its reduction in the presence of choline. 

(b) The Metabolism or Sulfonium Compounds 
(Dr. C. Wagner). The hydrolysis of sul- 
fonium compounds at neutral pH is associated 
with a standard free energy change of about 21,000 
calories. In an effort to investigate the possibility 
that cleavage of the sulfonium bond can be ener- 
getically coupled with cellular metabolism (as for 
example by the synthesis of ATP), an organism 
has been isolated from the soil that is capable of 
growing anaerobically with dimethyl-/?-propio- 
thetin as the major source of energy and carbon. 
Preliminary studies have been made to determine 
the nature of the fermentation process. It has 
been found that the decomposition of propiothetin 
is accompanied by the formation of propionic and 
acetic acids. 

The Metabolism of Isoprene Derivatives 

(a) Cholesterol Degradation. (Dr. M. G. 
Horning in collaboration with Prof. S. Bergstrom 
and Dr. H. Danielsson at the Karolinska Institute, 
Stockholm) . Incubation of human red blood cells 
with cholesterol-4-C 14 results in the formation of 
several degradation products which have been sep- 
arated into three major classes, acids, diols, and 
triols, by means of reverse phase chromatography. 
Although neither cholic acid nor chenodeoxycholic 
acid could be detected in the acid fraction, one of 
the acids produced appears to be identical with a 
compound formed when cholesterol is incubated 
with liver mitochondria. This compound may be 
a di- or tri-hydroxy coprostanic acid. 3/3,5a,6/3- 
trihydroxycholestane was identified as a compo- 
nent of the triol fraction and one of the diols was 
identified as 7/?-hydroxycholesterol. These results 
indicate that blood may have an important role 
in the metabolism of cholesterol. Future studies 
will be directed toward a more detailed analysis 
of this metabolic process at the enzyme level. 

(b) Isoprenoid Degradation (Dr. W. Seubert). 
In order to facilitate studies of the biochemistry 
of polyisoprene metabolism, an aerobic bacterium 
has been isolated from the soil that can utilize a 
simple diisoprene derivative, citronellol, as its sole 
carbon and energy source for growth. This or- 
ganism has been identified as a new species be- 



longing to the genus Pseudomonas and has been 
designated Pseudomonas citronellolis. In addi- 
tion to citronellol, higher analogues, such as 
farnesol and farnesoic acid, and the cyclic iso- 
prenoid, /8-ionone, are also utilized for growth. 
Results of various experiments to ascertain the 
mechanism of isoprenoid dissimilation have 
shown: (1) Citronellic acid accumulates in the 
culture medium as a transient intermediate dur- 
ing growth of the organism on citronellol; (2) In 
the presence of various isoprenoids, arsenite- 
inhibited resting cell suspensions catalyze the in- 
corporation of C 14 2 into acetate. This incorpo- 
ration does not occur in the absence of isoprenoids 
or when the isoprenoids are replaced by straight 
chain saturated fatty acids; (3) Incubation of 
arsenite-inhibited cell suspensions with C 14 2 and 
citronellic or farsenoic acids leads to the accumu- 
lation of /?-keto-acids ; (4) Cell-free extracts cata- 
lyze the incorporation of C 14 2 into acetate in the 
presence of either dimethylacrylyl CoA, gera- 
nionyl CoA or farnesyl CoA; (5) Extracts cata- 
lyze the incorporation of C 14 2 into acetoacetate 
in the presence of dimethylacrylyl CoA. These 
observations provide indirect support for the 
working hypothesis that the oxidation of polyiso- 
prenoids involves a stepwise degradation of the 
terminal isoprene moiety by reactions analogous 
to those involved in the oxidation of isovaleric 
acid. Thus the oxidation of farnesol would be 
visualized to occur by the following reaction 
sequence : 

I II III 

Farnesol-»farnesoic acid— ->farnesyl CoA— »gera- 

IV V 

nylacetoacetate— >geranionyl CoA->dimethylal- 

VI VII 

lylactoacetate-Klimethylacrylyl CoA-»acetoace- 

VIII 
tate— >2 acetyl CoA. 

According to this mechanism a fixation would 
occur at steps III, V, and VII. 

The Metabolism of Amino Acids 

(a) Threonine Biosynthesis (Dr. M. Flavin 
and Mr. C. Slaughter). The enzyme, threonine 
synthetase, catalyzes the conversion of 0-phospho- 
homoserine to threonine and orthophosphate. 
This novel reaction involves the elimination of an 
0-phosphoryl group from the alpha position of 



NATIONAL HEART INSTITUTE 



55 



homoserine and the simultaneous introduction of 
an hydroxyl group into the beta position. In a 
continuation of studies on the mechanism of this 
reaction, the enzyme has been purified 500-fold 
from Neurospora extracts and some of its proper- 
ties have been determined. Activity of the puri- 
fied enzyme requires the presence of added pyri- 
doxal phosphate. A further insight into the reac- 
tion mechanism has been obtained from studies 
with H 2 18 and D 2 carried out in collaboration 
with Dr. Tetsuro Kono of the McCollum-Pratt 
Institute. When the reaction is carried out in the 
presence of H 2 18 , O 18 is incorporated into thre- 
onine but not into phosphate. O-phosphothreo- 
nine is not decomposed. From these observations 
it must be concluded that the phosphate group of 
phosphohomoserine is removed through cleavage 
of the C-0 bond by an elimination reaction rather 
than by hydrolysis. When the reaction is carried 
out in the presence of 100 percent D 2 0, two atoms 
of deuterium are incorporated into threonine, one 
in the a-position. On the basis of these results it 
is tentatively proposed that threonine biosynthesis 
involves the intermediary formation of a Schiff 
base of vinylglycine and pyridoxal phosphate. 
The further observation that only 0.1 atom of 
solvent hydrogen per mole is incorporated into 
threonine when the reaction is carried out in 
H 3 2 0, indicates a high degree of discrimination 
against tritium. Tritium ions add to the postu- 
lated vinylglycine intermediate at only 2 to 3 per- 
cent of the rate of proton addition. 

(b) The Reductive Deamination of Glycine. 
(Dr. T. C. Stadtman) . Further studies have been 
made with the soluble enzyme system form 
Clostridium sticklandii that catalyzes the reduc- 
tion of glycine to acetate and ammonia by 1,3-di- 
mercaptopropanol, with the simultaneous esterifi- 
cation of orthophosphate to form ATP. At- 
tempts to purify the enzymes involved have re- 
vealed that a minimum of four proteins are essen- 
tial for catalysis of the overall reaction ; hence the 
mechanism is more complicated than was antici- 
pated. The conversion of glycine to acetate and 
the coupled phosphorylation are inhibited by an- 
timycine A. This and other indirect evidence 
supports the belief that a quinone derivative is in- 
volved. Analysis of the lipid fraction of C. stick- 
landii failed to detect the presence of tocopherols 
or quinones of the vitamin K or Coenzyme Q 



types. During the course of these experiments, it 
was found that the characteristic reddish orange 
color of extracts of C. stickla/ndii is due to the 
presence of remarkably high concentrations of 
the adenine vitamin B 12 coenzyme. Nutritional 
studies revealed that the concentration of the B 12 
coenzyme is a function of the rate of one-carbon 
metabolism by the cell. For example, supple- 
mentation of the culture medium with formate 
(which is fermentated largely to acetate) results 
in a much higher synthesis of the B 12 coenzyme. 
The potential significance of the B i2 coenzyme in 
one-carbon metabolism was further indicated by 
the discovery that two species of methane produc- 
ing bacteria are exceptionally rich sources of the 
coenzyme. No evidence could be obtained to in- 
dicate that the B i2 coenzyme is involved in the 
reduction of glycine to acetate. Evidence to the 
contrary was obtained by showing that two other 
strains of Clostridia capable of catalyzing the 
reduction of glycine to acetate, do not contain ap- 
preciable amounts of the B 12 coenzyme. 

(c) The Fermentation of y-Aminobutykic Acid 
(Mr. J. Hardman and Dr. T. C. Stadtman) . Pre- 
vious studies in this laboratory have shown that 
the fermentation of y-aminobutyrate by Clos- 
tridium aminobutyricum involves the interme- 
diary formation of succinic semialdehyde and 
y-hydroxybutyrate. The overall conversion of 
y-aminobutyrate to form y-hydroxybutyrate by 
cell- free extracts of this bacterium was found to 
be an oxidation-reduction process in which a- 
ketoglutarate and glutamate have catalytic roles. 
The reaction involves a transamination of the 
amino group of y-aminobutyrate to a-ketogluta- 
rate with the formation of succinic semialdehyde 
and glutamate; the latter compound then under- 
goes oxidative deamination by a DPN-specific de- 
hydrogenase to form a-ketoglutarate and DPNH. 
The oxidation of glutamate is finally coupled with 
the reduction of succinic semialdehyde to form 
y-hydroxybutyrate. The enzyme catalyzing the 
latter reaction has been purified about 20-fold 
from crude extracts. It is a DPN-specific dehy- 
drogenase showing marked substrate specificity. 
It appears to be a zinc-sulfhydryl enzyme. A re- 
quirement for two proximal sulfhydryl groups is 
suggested by the observation that the enzyme is 
inhibited by concentrations of arsenite that selec- 



56 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



tively react with disulf hydryl compounds. Aging 
of the purified enzyme results in a drastic loss of 
enzyme activity which is curiously restored by the 
addition of AMP. 

(d) Anaerobic Metabolism of the Dibasic 
Amino Acids, Ornithine and Lysine (Dr. V. 
Tarantola). Although ornithine was originally 
reported by Stickland and Woods, in their classic 
studies on the Stickland reaction, to undergo re- 
ductive deamination, it would appear that the 
mechanism does not involve a direct reductive de- 
amination. The final reduced and deaminated 
product, S-amino valerate, produced by extracts of 
Clostridium lentoputrescens, appears rather to be 
formed via a pathway involving a preliminary for- 
mation of a keto acid derivative of ornithine by a 
transamination followed by ring closure and re- 
duction to proline. A further reductive ring 
cleavage of proline by proline reductase and 1,3- 
dimercapto propanol yields S-amino valerate. 

A new Clostridium, as yet unidentified, has 
been isolated from swamp mud that is capable of 
growing on lysine as a single amino acid substrate. 
The products — ammonia, acetate, and butyrate — 
indicate that the organism probably catalyzes the 
same coupled oxido-reduction reaction originally 
discovered in C. stichlandii. The new organism 
is unique in that it can grow as a result of this 
fermentation and thus may be much more suitable 
as experimental material for study of this inter- 
esting cleavage of the lysine molecule. In C. stich- 
landii the formation of acetate and butyrate from 
lysine requires lipoic acid but the enzyme system 
proved to be very unstable. 

Laboratory of Chemistry of Natural 
Products 

The work of the Laboratory during the past 
year may be summarized in four categories: (1) 
studies in the structural chemistry of naturally 
occurring substances, particularly the Amaryllis 
alkaloid group, (2) the development of new meth- 
odology for lipid studies, and the application of 
new methods to lipid problems, (3) studies of the 
callicrein-callidinogen-callidin system, with par- 
ticular regard to isolation procedures and the de- 
velopment of assay methods, and (4) work on 
reactions related to biochemical transformations 
involving amine oxides and hemiacetals. 



(1) The work in structural chemistry con- 
tinued and extended past studies. A new ring 
system, not previously known to exist for either 
a synthetic or a natural substance, was found for 
the alkaloids montanine, coccinine, and manthine. 
The structural relationships between these com- 
pounds and other members of the Amaryllis alka- 
loid group was established through a transforma- 
tion linking the new series with another of the 
known groups. Since stereochemical relationships 
are of profound importance in determining the 
direction of biogenetic reactions, and usually lead 
to major variations in degree of physiological ac- 
tion, the previous structural studies were extended 
through the determination of the configuration 
and stereochemical relationships of the ethano- 
phenanthridine compounds. The C :D ring fusion 
is cis. Of interest is the fact that two groups of 
compounds of opposite stereochemical configura- 
tions occur in this field. These are based on the 
( + ) - and ( — ) -crinane series. A number of addi- 
tional structural determinations were made ; struc- 
tures were established for 1-acetyl-lycorine, epi- 
crinine, nerbowdine, 6-hydroxycrinamine, criwel- 
line, and haemanthamine. 

This work provides a considerable amount of 
new information about alkaloid structures, and 
about naturally occurring ring systems. The iso- 
lation work was mostly concerned with the new 
compounds discovered during the year, but atten- 
tion was also given to the problem of galanthamine 
isolation. This substance was introduced into 
Russian clinical medicine last year as a new and 
superior drug for the treatment of myasthenia 
gravis. Earlier chemical work in Russia led to 
the proposal of an incorrect structure for this com- 
pound ; the correct structure has since been estab- 
lished through work in Japan and in this labora- 
tory. Current studies have resulted in the devel- 
opment of a compound of still greater activity. 
Pharmacological studies of these substances are 
being carried out by Dr. R. L. Irwin (NINDB) 
and Dr. B. Holmstedt (Karolinska Institute, 
Stockholm) . 

Other alkaloid studies included those of the 
Limasia and Cassia compounds. Degradative 
studies of cassine were combined with instrumen- 
tal data to lead to a proposed structure; the sub- 
stance is a reduced pyridine compound with a 
long alkyl side chain. The Lunasia work was 



NATIONAL HEART INSTITUTE 



57 



terminated with several structural determinations. 

Present work in this category is directed toward 
completion of the Amaryllis structural studies, 
with particular regard to stereochemical prob- 
lems, and to the development of biosynthetic in- 
vestigations. 

(2) Until very recently most work in the lipid 
field was characterized by the use, out of necessity, 
of poor and inexact methods. This circumstance, 
more than any other, was responsible for the very 
slow rate of development in lipid chemistry and 
lipid metabolism over the last 30 years. The ap- 
parent relationship of lipid metabolism to athero- 
sclerosis, and the modification of human serum 
cholesterol levels by dietary fatty acids, has stimu- 
lated efforts to improve the methodology of lipid 
chemistry. In general, two broad areas of work 
were involved. The problem of identifying and 
measuring amounts of individual fatty acids was 
the more difficult of the two. Preliminary experi- 
ments showing that this could be done by gas- 
liquid chromatography were carried out in Eng- 
land in 1952, but 5 years later gas chromatographic 
methods were still ineffective for biological appli- 
cations. Preliminary studies indicated that the 
existing detection methods were not satisfactory 
and a continuing exchange of information with 
several U.S. laboratories was started in an effort 
to develop instruments and separation methods 
that would provide both the sensitivity and resolu- 
tion required for work with lipid. In this lab- 
oratory the characteristics of the argon detector 
were studied, and conditions for linearity defined. 
A study of liquid phases and of coating procedures 
was also carried out. Since the success of the 
method rests on the proper functioning of the 
column as well as the detection system, a new 
method for coating liquid phases was developed, 
and new procedures were worked out for prepar- 
ing polyester liquid phases with desirable separa- 
tion factors and high stability. Perhaps the most 
important factor involved in the use of polyester 
phases lies in the removal of exchange catalysts. 
From studies made in various ways it is believed 
that the polyester procedures developed here have 
solved this problem. 

The development of column chromatographic 
procedures for the separation of lipid classes has 
also been pursued. Existing methods were eval- 
uated and suitable modifications developed so that, 



by using silicic acid columns, the following classes 
can now be separated effectively: hydrocarbons, 
cholesterol esters, triglycerides, cholesterol, ceph- 
alins, lecithins, sphingomyelins, and lysolecithins 
(work in another laboratory has shown that mono- 
glycerides may also be separated, when present) . 

Several laboratory studies using these and re- 
lated methods are in progress. A detailed study 
of the lipids of severely atherosclerotic patients 
has been started. This work is in collaboration 
with Dr. Michael DeBakey (Baylor University) 
and Dr. B. G. Creech (Methodist Hospital, Hous- 
ton). The characterization extends to serum 
lipids, the lipids of the arterial block tissue, and 
adipose tissue. 

A method for the qualitative and quantitative 
estimation of the long chain base fraction of 
sphingolipids has been devised. This work dis- 
closed the presence of a new long chain base in 
human sphingomyelin. 

A comparison of the lipids (serum and aortic) 
of rabbits fed cholesterol with those of normal 
rabbits is in progress ; this is in collaboration with 
Dr. D. B. Zilversmit, who carried out the feeding 
experiments. 

Current work involves the continuation of meth- 
odological studies and the extension of new meth- 
ods to laboratory problems. The chief new area 
of methodology work is capillary chromatog- 
raphy. The highest degree of resolution obtained 
so far at 180-200° for fatty acid work is about 
50,000 theoretical plates. While this is far beyond 
that obtainable by any other method, it seems 
likely that a separating capacity of 300-500,000 
theoretical plates can be reached. Further, with 
this resolving power it should be possible to sepa- 
rate steroidal substances, if suitable liquid phases 
and modified techniques can be developed. This 
problem is under study in several laboratories. 
The extension to steroids is needed to study cho- 
lesterol formation and degradation more effec- 
tively. A high degree of resolution is also needed 
to separate positional and stereoisomers for un- 
saturated fatty acids. 

(3) The callicrein-callidinogen-callidin system 
has been studied with particular regard to isola- 
tion and assay procedures. This work was car- 
ried out in collaboration with Dr. S. J. Sarnoff 
and Dr. M. E. Webster. Methods were developed 
for the preparation of purified fractions of calli- 



58 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



crein (plasma, pancreatic and urinary) and for 
callidinogen. The objective is the characteriza- 
tion of these substances which may be of great 
importance in the functioning of the circulatory 
system. The problem is made difficult by the com- 
plexity of the protein and polypeptide mixtures 
which must be dealt with. A new assay method 
(Dr. Webster) is expected to be of considerable 
help. 

Current work is concerned with the perfecting 
of additional chromatographic purification meth- 
ods. 

(4) Amine oxide studies were resumed through 
an investigation of the catalyst requirements for 
the reaction. This work was started by Dr. J. C. 
Craig at the University of Sydney, Australia, and 
is now being continued at the same laboratory. A 
variety of iron complexes were used. It was found 
that both a-hydroxy and a-amino acids were ex- 
cellent complexing agents, and that the nature of 
the iron complex was important in determining the 
effectiveness of the rearrangement reaction. The 
data suggested that the mechanism involved a 
sequence of one electron shifts, with the transient 
formation of iron IV. The overall reaction leads 
to the removal of a methyl group from nitrogen, 
and it provides a model for the reaction of biologi- 
cal demethylation. 

A study of hemiacetal formation for long chain 
compounds was carried out. This work arose 
from observations on plasmalogens and related 
lipid substances in which an aldehyde reaction is 
clearly involved. It was found that long-chain 
aldehydes (palmitaldehyde) were highly reactive 
substances, and that both trimer formation and 
hemiacetal formation proceeded readily. The for- 
mation of esters from hemiacetals was also studied. 
These are models for studying lipid aldehyde reac- 
tions. Dr. Craig will continue this work at the 
University of Sydney. 



Laboratory of Chemical Pharmacology 

DEVELOPMENT OF NEW DRUGS 

Drugs for Arthritis, Gout, and Muscular Spasm — 
a Resume 

Largely because of studies in this laboratory 
with phenylbutazone and zoxazolamine, three 



derivative drugs for treatment of arthritis and 
gout and one for muscle spasm are now available. 
Two of these were introduced in the past year. 
The four drugs are: (1) Oxyphenbutazone (in 
arthritis), (2) Sulfinpyrazone (Anturan) (urico- 
suric agent), (3) Zoxazolamine (Flexin) (urico- 
suric agent), (4) Chlorzoxazone (Paraflex) (for 
treatment of muscle spasm). Two other potent 
phenylbutazone derivatives with prolonged urico- 
suric action, a para-methylsulfone derivative of 
phenylbutazone and a keto derivative of oxy- 
phenybutazone, may be of value as long acting 
uricosuric agents. 

Additional studies of phenylbutazone ana- 
logues confirm the view that these compounds act 
in the ionic forms and that an alkyl side chain 
is necessary for antirheumatic activity. Our con- 
tinuing search for a nontoxic antirheumatic agent 
will be guided accordingly. 

Reserpine Analogues 

Last year, studies with reserpine, in collabora- 
tion with Ciba Pharmaceuticals, indicated that 
the trimethoxybenzene ester linkage might be un- 
necessary for activity. A nonester reserpine has 
been found to release peripheral amines selec- 
tively without releasing brain amines. It is now 
in clinical trial for treatment of hypertension. 

BIOGENIC AMINES 

Drugs Acting Through Release of Brain Amines 

Whether the tranquilizing action of reserpine 
is due to loss of brain norepinephrine (NE) or is 
associated with inability of brain to bind sero- 
tonin (HT) is a crucial question. Dr. Brodie and 
his associates have shown that small doses of a 
reserpine analogue, dimethylaminobenzoyl meth- 
ylreserpate (Su 5171), deplete stores of brain NE 
considerably, without releasing stores of brain 
HT appreciably and without eliciting sedation. 
Larger doses of Su 5171 elicit sedation only if they 
affect 50 percent or more of brain serotonin bind- 
ing sites. 

Animals subjected to cold (or other stress), and 
then given reserpine, are not sedated; brain NE 
but not brain HT is depleted. In contrast to effect 
on reserpine, stress had no effect on chlorproma- 
zine action. Hypophysectomized rats, subjected 
to cold and then given reserpine, are sedated and 
brain HT stores are released, indicating that a 



NATIONAL HEART INSTITUTE 



59 



pituitary substance is needed for "stress" to pre- 
vent reserpine action. 

These results support the view, for which fur- 
ther pharmacologic evidence has been obtained, 
that "automatic" functions of brain are integrated 
by antagonistic neuronal systems : an adrenergic 
system (blocked by chlorpromazine) ; and a sero- 
tonergic system (stimulated by reserpine). If 
reserpine, as postulated, acts centrally by stimulat- 
ing a neuronal system (trophotropic) which inte- 
grates parasympathetic with somatomotor and 
psychic functions, then the drug should increase 
parasympathetic output from the CNS. Re- 
serpine elicits profuse salivation from the cannu- 
lated salivary gland, by a central parasympathetic 
action. Previous studies have shown that reser- 
pine-inducecl miosis and enhanced light reflex are 
a reflection of central parasympathetic stimula- 
tion. Since chlorpromazine does not increase cen- 
tral parasympathetic tone but instead decreases 
sympathetic tone, the two drugs must act central- 
ly on opposing autonomic systems. 

Drugs Acting Through Release of Peripheral NE 

Studies with syrosingopine (Singoserp) show 
that this compound can release peripheral NE, 
without releasing brain amines. This confirms the 
view that reserpine lowers sympathetic tone not 
by a central action, but by depleting peripheral 
NE and suggests that this drug may be useful in 
hypertension since it is likely to be effective in 
doses that do not produce depression. 

Studies with Guanethidine (Su 5864), a Ciba 
product, show that the drug probably lowers 
blood pressure by depleting peripheral NE but 
not brain NE. It presumably acts by a different 
mechanism than does reserpine since it does not re- 
lease serotonin, and releases NE more slowly than 
does reserpine. 

Drugs Acting Through Blocking the Metabolism 
of Amines (Monoamine Oxidase (MAO) 
Inhibitors ) 

Toxicity — Certain MAO inhibitors, especially hy- 
drazine derivatives of phenylethylamine, produce, 
in dogs, degenerative lesions in the inferior olivary 
nucleus and in the pyriform lobe accompanied 
by neurological symptoms. JB 516 (Catron) pro- 
duces such effects in dosage as low as 0.5 mg per 

556044—60 6 



kg daily. Preliminary data suggest similar re- 
sults in cats, but not in rabbits and monkeys. 

Effect on Brain Amines. — Previous studies using 
MAO inhibitors have established a rapid turnover 
for brain HT but a slow turnover for NE ; how- 
ever, excitation elicited by MAO inhibitors is tem- 
porally related to rise in brain NE. Turnover of 
dopamine has also been found to be extremely 
rapid (50% in 15 minutes), indicating that MAO 
is important in the metabolism of this catechol- 
amine in brain. 

Further evidence favors the view that the chief 
role of MAO is not to metabolize physiologically 
released NE, but to regulate amounts of NE and 
HT stored in neurons. Thus, the two enzymes, 
O-methyltransferase and MAO, may have quite 
different roles, the former modifying released 
catecholamines, the latter regulating the amount 
in storage. 

Mechanism of Action of the Anti-depressant 
Drug, Imipramine (Tofranil) 

This drug, structurally related to chlor- 
promazine, is an antidepressant but is not a MAO 
inhibitor. It does not elicit excitation in normal 
man or in animals, but acts only in the depressed 
individual. Studies in this laboratory have 
shown that Tofranil blocks a number of the cen- 
tral actions of reserpine (but not of chlor- 
promazine). It blocks the ability of reserpine to 
potentiate alcohol and barbiturates in mice and 
rats and to produce sedation, ptosis, and miosis in 
rats. It does not, however, interfere with release 
of brain amines by reserpine. Preliminary evi- 
dence indicates that chlorpromazine, despite its 
depressant action, also has a delayed action in 
blocking reserpine. These findings are tentatively 
explained by the working hypothesis that pheno- 
thiazines and related compounds may have two 
actions: central adrenergic blocking and central 
serotonin blocking. Chlorpromazine exerts both 
effects, with antiadrenergic predominating. To- 
franil exerts both actions with antiserotonin pre- 
dominating. 

Mechanism of Uptake of Catecholamines by 
Brain Tissue 

In previous studies it has been shown that plate- 
lets take up HT and catecholamines through active 



60 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



transport, by a mechanism blocked by reserpine. 
Similar studies are being undertaken to ascertain 
whether the uptake of amines by brain also in- 
volves active transport. On incubation in vitro 
with plasma, brain slices including hypothalamus, 
thalamus, rhinencephalon, and pituitary, but not 
cerebellum, take up epinephrine. In contrast, 
brain slices from animals pretreated with reser- 
pine do not concentrate epinephrine. 

Studies on Distribution and Role of NE, HT, and 
Their Synthetic Enzymes in Nervous Tissue 

(a) Micromethods for the estimation of 0.04 y 
of NE and 0.20 y of HT were developed. 

(b) The constancy of the ratio of 5HTP to 
DOPA decarboxylase activities throughout the 
cat brain indicates the same decarboxylase acts on 
both substrates. The concentration of amines and 
enzymes is low not only in cerebellum and cortex 
but also in those sensory nuclei which are situated 
in the brain stem. In contrast, they are high only 
in those parts of brain associated with "automatic 
behavior," e.g., reticular formation, hypothalamus, 
and rhinencephalon. 

(c) Levels of brain amines are being related to 
gross behavior and drug action at various ages. 
At birth the level of NE in rats is only about 20 
percent of adult level, while the HT level is about 
40 percent adult level. The levels increase with 
age and the results suggest an association of brain 
amine levels and development of behavioral pat- 
terns. Studies of guinea pigs are underway, since 
these animals are born more fully developed than 
rats. 

(d) Reserpine decreases by 90 percent the NE 
content of the superior cervical ganglion in cats. 
The post-ganglionic response to a preganglionic 
electrical stimulation is markedly enhanced. This 
suggests that normally NE may have a role in the 
ganglionic response to acetylcholine. 

Histamine Studies 

The development of a specific and simple fluoro- 
metric procedure for histamine assay in tissues 
should help in studies of this substance whose 
physiological role and biosynthesis are still un- 
known. Application of the method to brain has 
shown that contrary to many reports, there is little 
histamine present and this may be associated with 



non-nervous vascular tissue. By use of com- 
pounds that inhibit MAO but not diamine oxidase, 
it has been shown that MAO has no role in metab- 
olizing histamine in vitro and presumably in vivo. 
It has also been found that the reserpine releases 
histamine from rabbit platelets but from no other 
tissue. Evidence indicates that this release may 
be mediated by free HT. 

PASSAGE OF DRUGS ACROSS MEMBRANES 

Blood-Brain Barrier 

Kinetic data from a study of the penetration 
into CSF of 20 drugs with diverse structures and 
physical properties now provide considerable con- 
fidence in the assumption that the blood-brain 
barrier acts as an inert lipoid boundary to drugs. 
Only the undissociated forms of the drugs appre- 
ciably penetrate the CSF and at rates determined 
by their heptane/ water partition ratios. How- 
ever, certain parts of brain, including both lobes 
of the pituitary, the intercolumnar tubercle and 
the area postrema have no blood-brain barrier to 
N-acetyl-4-aminoantipyrine, sulfaguanidine, ra- 
dioactive sodium, and labelled epinephrine. In 
contrast to their slow passage into CSF, water- 
soluble substances such as sucrose and phenol red 
readily leave the CSF. They may leave via the 
arachnoid villi and the rate may be related to the 
turnover of CSF. 

Penetration of Drugs into Cells 

The penetration of a number of drugs from 
plasma into red cells has been studied. Sub- 
stances generally cross this boundary much more 
rapidly than blood-intestinal or blood-brain bar- 
rier perhaps because of large surface/volume re- 
lationship. The relative rates of entry of organic 
bases seem to be determined by lipid solubility, 
but the passage of organic acids does not fit the 
lipoid barrier pattern. Instead, organic electro- 
lytes follow a pattern not unlike that shown by 
the usual mineral ions. Thus, sulfonic acids 
penetrate many times faster than quaternary am- 
monium ions, and acids at the steady state may 
occupy only a fraction of total volume of cell 
water in contrast to bases which occupy the total 
volume of cell water. 



NATIONAL HEART INSTITUTE 



61 



Passage of Purines and Pyrimidines Across the 
Intestinal Tract 

A transport mechanism for the absorption of 
uracil and thymine is present in the intestinal 
mucosa. Active transport has been shown in 
vitro using everted intestinal sacs. The mecha- 
nism requires oxygen and is saturated at low con- 
centrations of pyrimidine. Thymine transport is 
blocked by uracil, hypoxanthine and other pyrim- 
idines and purines. 

DRUG METABOLISM 

Substances (Antimetabolites) Metabolized by 
Relatively Specific Enzymes of Intermediary 
Metabolism 

Studies of the metabolism and mechanism of 
action of the anti-tumor agent, 6-chloropurine, 
have been continued. Last year the isolation of a 
new substance, 6-chlorouric acid, was reported. 
Studies this year show that the purine skeleton of 
6-chloropurine is incorporated into the adenine 
and guanine of both ENA and DNA. Further- 
more, 6-chloropurine inhibits the turnover of both 
ENA and DNA, as followed by p 32 in vivo, in 
liver slices, and in isolated liver nuclei. 

Substances Acted on by Extremely Nonspecific 
Enzymes Not Involved in Intermediary 
Metabolism 

Further studies have been made on the oxidative 
enzymes in liver microsomes. 

(a) Microsomal Sulfoxidasc. The enzyme sys- 
tem that oxidizes 4,4'-diaminodiphenyl sulfide and 
chlorpromazine to the corresponding sulfoxides 
has been definitely characterized as another micro- 
somal enzyme (a sulf oxidase) requiring TPNH 
and 2 . 

(b) Nicotine. The key step in oxidation of nico- 
tine is not cotinine, as suggested in last report, but 
hydroxylation of the carbon atom next to nitrogen 
in side chain to yield a cyclic aldehyde (hydrox- 
ynicotine) . This supports the view that the first 
step in demethylation is hydroxylation of the 
methyl group. Elucidation of this reaction may 
indicate how ring systems are split in the body. 

(c) Mechanism of Microsomal Drug Oxidation. 
This mechanism involves direct utilization of oxy- 



gen and may share the same donor of "active" 
oxygen as cholesterol. A similar mechanism may 
be involved in the hydroxylation of steroid rings 
to form corticoids. In previous work it was shown 
that TPNH is oxidized in the absence of drug to 
yield H 2 2 . Two drugs, 4,4'-diaminodiphenyl 
sulfide and p-ethoxyacetanilide, do not affect the 
rate of TPNH oxidation, but cause a decrease in 
formation of H 2 2 equivalent to the formation of 
their metabolites. This supports the view that 
TPNH and 2 react in microsomes to form a 
"hydroxyl donor." In the absence of drug sub- 
strate, a part of the donor participates in normal 
hydroxylation reactions (e.g., cholesterol forma- 
tion) , and the rest breaks down to H 2 2 . In the 
presence of drug, a part of the "hydroxyl donor" 
is used by a number of non-specific "drug en- 
zymes" to hydroxylate foreign compounds. 

(d) Induced Enzyme Formation. Administra- 
tion of certain drugs, e.g., phenylbutazone, amino- 
pyrine, 3-4,benzpyrene, phenobarbital, increases 
the ability of rats to metabolize the same or closely 
related drugs. This increased activity is also 
shown in vitro by liver microsomes. This effect 
may explain, in part, the tolerance to barbiturates. 
A particularly interesting observation is that bar- 
biturates lower coumarin anticoagulant levels in 
man. The tolerance of hyperthyroid subjects to 
drugs prompted a study of effects of thyroxin on 
drug metabolism. Pretreatment of rats with 
thyroxin decreases the duration of zoxazolamine 
paralysis and increases the activity of liver micro- 
somal enzyme that metabolizes the drug. 

(e) Biochemical Evolution. It was shown in 
previous studies that the appearance of drug 
metabolizing enzymes is associated with adaption 
to living on dry land. In continuing studies of 
the ontogenetic development of drug enzymes, we 
find that liver microsomes in chicken embryos, un- 
like those in the mammalian embryo and the tad- 
pole, contain enzymes that oxidize drugs and 
which require TPNH and 2 . The possession of 
these enzymes by the chicken embryo may be re- 
lated to its nonaqueous milieu. 

STUDIES IN BIOCHEMICAL BEHAVIOR 

Preliminary experiments in rats on the tem- 
poral relation between pituitary stimulation and 
various responses were undertaken. Typical 



62 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



stimuli were cold, intradermal formaldehyde, 
ethionine, reserpine, ethanol, dibenamine, carbon 
tetrachloride, 3-methylcholanthrene and Tofranil. 
(No responses are obtained in hypophysectomized 
animals.) (1) Adrenal ascorbic acid is rapidly 
reduced, returning to normal in about 5 hours. 
With persistent stimuli (cold and methylcholan- 
threne), adrenal ascorbic level returns to higher 
than normal value. This probably reflects the 
induced, increased synthesis of ascorbic acid in 
the rat, since in guinea pigs which do not make 
ascorbic acid, the adrenal levels remain low in 
persistent stress. (2) Plasma corticosteroid levels 
are maximal in 1 to 4 hours and then return to 
normal. With persistent stress, the high levels 
are maintained. (3) Plasma-free fatty acids 
(FFA) usually are maximal in 1 to 4 hours and 
then return to normal, except in persistent stress. 
Anomalous results are obtained with electroshock 
which lowers FFA and with Tofranil which pro- 
duces a delayed response. (4) Although many 
stimuli cause a rise in FFA only some of them 
result in deposition of liver triglyceride. This 
suggests that something must also happen in liver 
to cause fatty deposition. (5) The activity of 
tryptophane peroxidase (TPO) in liver is in- 
creased by all stimuli. (6) A chemical picture 
typical of "stress" is elicited by the "antistress" 
compounds, reserpine and chlorpromazine. 

Specific Studies 

(a) Reserpine. It has been reported that reser- 
pine pre treatment inhibits the responsiveness of 
the pituitary-adrenal axis to stressful stimuli. 
Surprisingly, reserpine depletes adrenal ascorbic 
acid in the rat, increases the level of circulating 
corticoids and increases the level of liver trypto- 
phane peroxidase. No effects are observed in hy- 
pophysectomized rats. Preliminary results indi- 
cate that the effect on pituitary may be related to 
change in brain amines. 

(b) 3-Methylcholanthrene. This compound in 
rats causes prolonged pituitary stimulation lasting 
up to 48 hours. The effects are depletion of adre- 
nal ascorbic acid, increase in plasma corti- 
costeroids, increase in plasma FFA, increase in 
liver TPO and antagonism of reserpine sedation. 
Other carcinogenic agents do not elicit this marked 
response. 3-Methylcholanthrene affected the 



pituitary-adrenal axis in doses as low as 0.1 /*g/kg. 
The mechanism of this effect is under study. 

(c) Effect of Various Pituitary Stimuli on 
Various Body Enzymes. The most sensitive index 
of pituitary stimulation found thus far is the in- 
crease in liver TPO. Not only is TPO increased 
by stimuli previously mentioned but by certain 
barbiturates. None of the stimuli is effective in 
hypophysectomized animals. Of interest is the 
finding that tryptophane in doses said to act as 
specific inducer of TPO, also decreases adrenal 
ascorbic acid, and increases plasma corticoids and 
FFA. 

The effect of many drugs in stimulating the bio- 
synthesis of L-ascorbic acid in rats, and which has 
been shown in this laboratory to result from a 
stimulation of glucose metabolism via glucuronic 
acid, 1-gulonic, etc., is also a pituitary response, 
which occurs in adrenalectomized rats, but not in 
hypophysectomized animals. Drugs that increase 
ascorbic acid synthesis also increase the activity of 
liver microsomal enzymes. Whether this effect on 
liver microsomes is an adaptive response to reduce 
toxicity to foreign compounds or is a more gen- 
eralized response remains to be seen. Other en- 
zymes that are stimulated by drugs are those in 
liver that convert galactose to glucuronic acid. 

(d) Effect of Drugs on Triglyceride Mobiliza- 
tion' and Deposition. Triglyceride deposition in 
rat liver is produced by CC1 4 , ethanol, and ethio- 
nine. The fatty acids appearing in the neutral 
fat of liver appear to be mobilized from fat depots 
(collaboration with Dr. Marjorie Horning), but 
ethanol may also stimulate their synthesis in liver. 
Triglyceride deposition may be blocked by large 
closes of adrenergc blocking agents. 

Isolation of Cardiotonic Substances from Mam- 
malian Tissues 

In addition to lysolecithin, previously reported, 
other active factors, all acidic lipids, have been 
obtained from mammalian tissues. These include : 

(1) Beef blood factor — probably cis vaccenic acid. 

(2) Beef heart factor — an acidic factor which, 
from infrared spectra, may be a lactonized hy- 
droxy fatty acid. (3) Rabbit serum factor; infra- 
red also suggests that this is an unsaturated lac- 
tonized hydroxy acid. 



NATIONAL HEART INSTITUTE 



63 



It is believed that these substances may influence 
the passage of ions through membranes, as a re- 
sult of which the muscle contractility may be 
increased. 

STUDIES WITH ASCORBIC ACID 

Ascorbic acid is synthesized in rats from glu- 
cose through D-glucuronic acid and L-gulonic 
acid. During the past year evidence has been 
obtained for an enzyme system in rat liver which 
synthesizes D-glucuronic acid through uridine 
nucleotide precursors. The occurrence of such re- 
actions in the intact animal was confirmed by ex- 
periments showing that D-galactose is a consider- 
ably better precursor of L-ascorbic acid than is 
D-glucose. 

Information has also been obtained on the me- 
tabolism of L-ascorbic acid. An enzyme system 
in guinea pig and rat tissues which decarboxylates 
L-ascorbic acid has been purified and two sugar 
acids, L-lyxonic and L-xylonic acids, have been 
identified as products of the reaction. A strain 
of yeast has been adapted to grow on L-ascorbic 
acid as its sole carbon source ; this observation may 
furnish a powerful tool for studying the mecha- 
nisms involved in ascorbic acid metabolism. 



Laboratory of Technical Development 

GAS CHROMATOGRAPHY 

Considerable effort has been devoted to the de- 
velopment of methods of increasing the sensi- 
tivity and versatility of gas-chromatography 
techniques. The radiofrequency (RF) discharge 
detector system has been thoroughly evaluated 
and shown to be applicable to both standard col- 
umns and capillary columns and to provide 
high sensitivity. Comparisons of the RF detec- 
tor with argon ionization detectors showed an ap- 
proximately 10-fold greater sensitivity for the RF 
discharge detector. Variations in conditions pro- 
duce changes in the baseline with the RF detec- 
tor, whereas similar variations yield, with the 
argon detector, changes in sensitivity with con- 
sequent errors in the size of the peaks but with a 
stable baseline. This makes the records produced 
with the argon detector superficially more attrac- 



tive since the errors are less obvious. At present 
it is simpler to use the argon detector but im- 
provements in the radiofrequency excitation 
source may change this. Studies of the radiofre- 
quency discharge as a source of ionization which 
can be measured downstream from the gas dis- 
charge and to improve the radiofrequency exci- 
tation systems are continuing. 

The need for a satisfactory system for the ra- 
dioassay of C-14-labeled compounds was pointed 
out by the Committee on Lipid Analysis and a 
program was undertaken to solve this problem. 
The method developed consists of capturing the 
effluent in short sections of column using anthra- 
cene crystals coated with one of several liquid 
phases shown not to interfere with the counting. 
These short columns permitted direct radioassay 
by the usual automatic scintillation counting tech- 
niques. The efficiency of this system is very close 
to theoretical limits. In addition to the above 
noted gas chromatographic techniques, Dr. 
Karmen participated in several studies in coopera- 
tion with other laboratories, notably in the studies 
on chylomicron composition after fat ingestion 
and a study of binding of unesterified fatty acids 
to various serum proteins with Drs. Bragdon and 
Shafir, respectively. An exploration of the appli- 
cation of zeolite molecular sieves to analysis of 
respiratory gases was undertaken as a project for 
one of the high school teachers on summer train- 
ing. Although the attempt to produce a zeolite 
sieve to separate all of the respiratory gases in 
one step was not successful, the study suggested 
several other potential uses of these sieves. 

Since it would be particularly advantageous to 
have a detector for gas chromatography columns 
that would not only be sensitive but would also 
give information as to the molecular weight of the 
gas, a gas chromatographic detector, based on 
the measurement of sound velocity, was developed. 
From a consideration of the parameters contribut- 
ing to the change in velocity, it would appear 
that if the quantity were known an estimate of the 
molecular weight could be obtained from the sonic 
velocity curve. The sensitivity of ordinary meth- 
ods of sound velocity measui-ement suggested that 
the method would be of no use unless some high 
sensitivity system for sound velocity measurement 
were developed. An ingenious electronic-circuit 



64 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



system, far exceeding expectations as to sensitiv- 
ity, was devised by Mr. Noble. The method of 
frequency multiplication and phase comparison 
available with this apparatus makes possible sensi- 
tivities which can be utilized only if suitably stable 
sample chambers can be developed. Test runs 
using this sound velocity system showed it to 
possess sensitivity exceeding that of the thermal 
conductivity system. The relative complexity of 
the electronic equipment and the requirement for a 
split stream differential system can be matched by 
an expectation of very high sensitivity and an ex- 
tremely small cell volume. The system in current 
operation has a cell volume of 0.08 ml and this 
can be reduced several fold without sacrifice of 
capability. This equipment in its present form, 
with a Linde sieve type 13X, can yield an analysis 
of 5 /A of air for oxygen and nitrogen content in 
0.4 minutes. 

MICROANALYSIS BY EXCITATION IN LOW 
PRESSURE HELIUM DISCHARGE 

Several aspects of the helium discharge and its 
ability to excite spectral emission were investi- 
gated. It was found that the discharge would 
excite molecular and atomic emission from volatile 
organic compounds. This suggested the possi- 
bility of spectral analysis of the glow as the frac- 
tions appeared from the chromatograph column. 
The emission spectra were taken and studied for 
several types of compounds. The complexity of 
the spectra produced by mixtures of gases does 
not encourage specific analysis by this means. 
Since spectral analysis showed a relatively clear 
zone above 500 millimicrons, it was concluded that 
organic materials in blood and urine specimens 
would not produce interfering bands that would 
overlap the region used for determination of the 
alkali metals. The work on emission spectro- 
scopy by means of helium discharge for the de- 
termination of alkali metals is now continuing 
after a period during which the apparatus was 
modified and these spectra explored. The ap- 
paratus is now improved with stabilized methods 
of measurement, stable excitation sources and a 
new chamber allowing volatilzation of the sample 
on a heated platinum wire. In addition, prepara- 
tion of measuring pipettes has been facilitated by 
the use of a commercially available quartz tubing 



33 (I I.D. available under the name of Santo tube 
from Monsanto. 

ERRORS IN CATHETER-RECORDING SYS- 
TEMS AND THEIR CORRECTION 

The completion of Mr. Noble's work on a precise 
variable frequency hydraulic pressure generator 
has made possible accurate analysis of the per- 
formance of various intracardiac pressure-measur- 
ing systems. It has been shown that pressure 
curves taken by cardiac catheterization, as normal- 
ly performed, are distorted in shape and time. 
As these errors may be wrongly interpreted as due 
to the vascular system rather than the instru- 
ments, several efforts have been made to develop 
a catheter-tip transducer. A solution developed 
by Mr. Noble makes it possible to compensate elec- 
trically for the errors introduced by the catheter. 
The simple electronic circuit can be adjusted for 
the specific catheter so that all the errors due to 
aging of the catheter and the particular pressure 
transducer used are compensated at once. Tests 
of the efficacy of the system are being run in 
collaboration with Dr. Guy Barnett of the Section 
on Cardiodynamics. The current method utilizes 
the recently developed hydraulic pressure genera- 
tor to make and confirm the compensation, but a 
relatively simply method of setting the compen- 
sator can be developed using a simple, transient 
pressure pulse applied to the catheter after it is 
in place. 

COMPUTER FOR ANALYSIS OF OVERLAP- 
PING DISTRIBUTIONS 

The analog computer for analysis of overlap- 
ping distribution functions was completed this 
year and tested for ability to resolve overlapping 
peaks. The instrument's utility will be more com- 
pletely evaluated by application to resolution of 
absorption spectra by Dr. Hayes, who has pre- 
pared and purified for this purpose some specific 
compounds with known absorption spectra. In 
addition, the application of the instrument to the 
detailed analysis of infrared spectra is anticipated. 

EVALUATION AND QUANTIFICATION OF 
VALVULAR REGURGITATION 

An investigation of methods of quantitating 
mitral valve regurgitation has been undertaken 



NATIONAL HEART INSTITUTE 



65 



by Dr. Peter Frommer in collaboration with the 
Cardiology Section of the Surgery Branch. 
Methods have been reviewed and the possibilities 
of the dye dilution technique utilizing radio- 
opaque dye and serial X-ray films are under con- 
sideration. The exploration has been facilitated 
by the use of a simple analog computer which pro- 
vided easily changed parameters with idealized 
curves produced to indicate the effects of backflow 
and dilution. From the examination of the prop- 
erties of the analog system, it would appear that 
the radio-opaque dye dilution technique has 
promise but puts some stringent requirements on 
the instrumentation and clinical procedures. The 
analog system proved so convenient and informa- 
tive in the evaluation of partial mixing and re- 
gurgitation that a paper on the method is in 
preparation. 

FLOW METER DEVELOPMENT 

Dr. Frommer has guided the investigation of 
the possibility of utilizing for flow measurement 
the Coriolis force produced when a fluid is made 
to gain and lose angular momentum as it passes 
through a rotating loop of pipe. This type of flow 
meter would measure actual mass flow, be inde- 
pendent of viscosity and provide a convenient 
form of differential flow meter. A rather large 
model system was constructed and was sufficiently 
promising to encourage further work on more 
compact designs. This was a summer project al- 
most entirely performed by a summer student 
employee. 

Investigation of nuclear magnetic resonance 
phenomena in the measurement of blood flow and 
their potentialities in analytical instrumentation 
is continuing. Mr. Kudravcev has constructed 
several types of nuclear resonance apparatus and 
circuits have been developed which yield a con- 
siderable reduction in the size and in the instabil- 
ity noted in previous equipment. It still appears 
feasible to evolve a flow meter system sensitive to 
volume flow using nuclear resonance principles. 
The possibility that such a device can be made 
without requiring intimate electrical contact may 
compensate for the complexity of the apparatus. 
Application to pulsating flows has not yet been 
minutely examined. If the volume flow measure- 
ment should prove impractical, the velocity tech- 



nique may have value. Considerable information 
has been gained with regard to construction of 
simplified nuclear magnetic resonance systems and 
this will presumably be useful in the development 
of analytical devices should development of a 
practical flow meter prove impossible. The ana- 
lytical characteristic of the system may also be 
applied to flow measurement as a method for de- 
termining the concentration of a nuclear "dye" 
as it goes past the detector. Fluorine, for ex- 
ample, is easily detected and could be used with 
present apparatus in a dye dilution system. 
Highly fluorinated materials such as freon could 
probably be utilized. 

THEORETICAL ANALYSIS OF TRANSPORT 

Dr. Stephenson has evolved several contribu- 
tions to the theoretical analysis of transport in 
biological systems, and this work has resulted in 
two papers on the subject in the Bulletin, of Mathe- 
matical Biophysics. This theoretical material has 
been helpful in the analysis of Dr. Frederickson's 
experiments on fatty acid metabolism. The data 
have been analyzed and programmed for computa- 
tion on the IBM. The investigation of the physics 
of the ultrarapid freezing of water in biological 
materials is continuing. A fuller understanding 
of the process may lead to information on the 
structure of biological material and aid in the 
design of methods for preservation of material by 
freezing and drying, both for banking of biological 
materials and the preparation of material for elec- 
tron microscopy. 

PHOSPHORESCENCE ANALYSIS 

An investigation of the application of phospho- 
rescence to the analysis and characterization of 
biological materials is being pursued by Dr. Hayes 
with the loan of the phosphorimeter from the 
American Instrument Company. One of the first 
problems was to find a solvent that would be suit- 
able for investigation of water soluble materials. 
A survey of materials that would form a satis- 
factory glass at a temperature at which phospho- 
rescence observations could be made was under- 
taken, and it was found that propylene glycol at 
minus 80° formed a satisfactory glass. At the 
temperature of liquid nitrogen, however, the 



66 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



solvent would crystallize and spoil the optical 
properties of the system. Accordingly, liquid 
gases for the cooling of the sample were surveyed 
to find one which would provide temperatures be- 
tween those avilable from carbon dioxide and 
liquid nitrogen. Nitric oxide was found satisfac- 
tory. Utilizing the new system, a survey of bio- 
logical compounds of biological interest is cur- 
rently in progress. Evaluation of phos- 
phorimetry as a method for analysis and charac- 
terization of materials will be pursued. 

ULTRASONICS 

Dr. Weissler's investigation of the effects of 
ultrasonic irradiation of hemoglobin has shown 
that the products of ultrasonic irradiation of 
water participate in the destruction or conversion 
of the hemoglobin molecule, and that additives 
in the form of dissolved gases or chemical agents 
modify the form of degradation. This work sug- 
gests the possibility of modifying the destructive 
effects of ultrasonic irradiaion when utilized for 
liberation of biological materials from cells and 
tissues by high powered ultrasonic disintegration. 
In addition, the selection of the environment may 
provide for selective isolation of particular en- 
zymes by protecting one at the expense of the 
other. 



Laboratory of Cardiovascular Physiology 

STUDIES ON THE HEART 

The Diastolic Pressure — Myocardial Segment 
Length Relation in the Ventricle. Observations 
on the Contribution of Atrial Systole 

The relation between left ventricular diastolic 
pressure and the simultaneously recorded Changes 
in the length of a segment of left ventricular my- 
ocardium was intensively studied. The shape of 
this curve laid the basis for a clearer understand- 
ing of those circumstances under which atrial sys- 
tole will produce its greatest contribution to the 
elongation of the fibers of the ventricular myo- 
cardium. Further, when considered together 
with the curve relating pressure to stroke work 



it gave added support to the position that, in any 
given metabolic state, the force of contraction 
of the ventricle is a function of the fiber length 
from which the contraction begins. These in- 
vestigations show the importance of atrial systole 
for ventricular filling. 

The Influence of Cardiac Sympathetic and Vagal 
Nerve Stimulation on the Relation Between Left 
Ventricular Diastolic Pressure and Myocardial 
Segment Length 

The full curve relating ventricular pressure to 
myocardial segment length showed clearly the 
lack of any change in this relation with sympa- 
thetic or vagal stimulation. These observations 
made it clear that the family of curves relating 
filling pressure to stroke work is accompanied by 
a family of curves relating initial fiber length to 
stroke work. A corollary of these data is that, 
under sympathetic stimulation, the ventricle will 
produce more external work from any given ini- 
tial fiber length as well as from any given end- 
diastolic pressure. This constitutes a substantial 
advance. 

In the course of these studies it was found that 
tachycardia, without concomitant sympathetic 
stimulation, so impinges on diastole (especially 
if the stroke volume is high) that the ventricle 
does not have sufficient time to acquire its "nor- 
mal" diastolic pressure-length relation. The ad- 
dition of sympathetic stimulation at the same 
heart rate so condenses systole that it permits the 
same ample diastole that would have occurred at 
a lower heart rate. The technical and concep- 
tual advances in these studies have helped to 
bring into clearer focus the importance of the 
simultaneous positive inotropic effect on the ven- 
tricle when tachycardia is induced by the sympa- 
thetic outflow. 

The Regulation of the Ventricle's Contraction: 
The Influence of Cardiac Sympathetic and Vagal 
Nerve Stimulation on Atrial and Ventricular 
Dynamics 

With the advances described above it was possi- 
ble to systematize understanding of the means by 
which the central nervous system can produce 
acute changes in the performance of the heart 
other than by the well known effects on rate. 



NATIONAL HEART INSTITUTE 



67 



The pertinent observations may be stated as 
follows : 

1. At constant heart rates efferent stimulation 
of the vagus nerve exerts a profound depressant 
effect on the strength of the atrial contraction and 
can thereby influence ventricular filling and ven- 
tricular stroke work ; mean atrial and thus venous 
pressure are elevated at any given level of cardiac 
work or cardiac output during vagal stimulation 
despite the fact that the vagal stimulation used 
does not alter the performance characteristics of 
the ventricle. The effects of vagal stimulation 
are blocked by atropine. 

2. Stellate ganglion stimulation or norepine- 
phrine infusion augments the strength of atrial 
contraction and thus the atrial contribution to ven- 
tricular filling. The augmented atrial contraction 
takes place in a shorter period of time. 

3. Stellate ganglion stimulation or norepine- 
phrine infusion increases the external work and 
power produced by the ventricle from any given 
filling pressure and fiber length. 

4. There is a family of curves representing the 
relation between end diastolic fiber length and 
stroke work as well as a family of curves repre- 
senting the relation between filling pressure and 
stroke work. 

5. When taken together with the well known 
sympathetic and parasympathetic effects on heart 
rate, the above data are believed to comprise a 
reasonably comprehensive description of the means 
available to the central nervous system for directly 
inducing acute changes in the activity of the heart. 

On the basis of these observations, Dr. Sarnoff 
and his colleagues propose what they refer to as 
"the law of the innervated heart" as follows : 

1. If the effective catechol amine stimulus re- 
mains constant, the contraction of the ventricle 
varies with its end diastolic pressure and fiber 
length. If the end diastolic pressure and fiber 
length remain constant, the contraction of the 
ventricle varies with the effective catechol amine 
stimulus. 

2. The central nervous system has available ef- 
ferent neuronal pathways to the heart by means 
of which it can vary ventricular end diastolic pres- 
sure and fiber length while keeping the effective 
catechol amine stimulus constant, means by which 
it can increase the effective catechol amine stimu- 
lus, or both. 



The Regulation of Ventricular Contraction by the 
Carotid Sinus: Its Effect on Atrial and Ventricu- 
lar Dynamics 

The role of carotid sinus baroreceptors in cir- 
culatory regulation was reevaluated and showed 
that a dominant aspect of the carotid sinus regula- 
tory activity is to augment or diminish the con- 
traction of the ventricle. The basis for this con- 
clusion is as follows : 

1. Carotid hypotension diminishes venous dis- 
tensibility. The net effect of such a change, if it 
alone occurs is an increased ventricular end di- 
astolic pressure and fiber length and thus an aug- 
mented ventricular contraction. Splenic contrac- 
tion would have the same effect. 

2. Carotid hypotension augments and shortens 
the atrial contraction. The net effect of such an 
atrial augmentation, if it alone occurs, is an in- 
creased ventricular end diastolic pressure and 
fiber length and thus an augmented ventricular 
contraction. 

3. Carotid hypotension directly augments the 
work produced by the ventricle from any given 
end diastolic pressure or fiber length and pro- 
duces more complete systolic emptying. 

4. Carotid hypotension augments ventricular 
power as well as contractility, since it shortens the 
systolic time for any given amount of work pro- 
duced, provides more rapid relaxation, and thus 
diminishes filling impedance. If this alone occurs, 
it provides for a. longer interval of diastolic filling 
than would otherwise occur and thus produces an 
augmented ventricular contraction, a factor which 
becomes especially important at high heart rates. 

5. The catechol amines secreted by the adrenal 
medulla in response to a lowering of carotid sinus 
pressure would be expected to reenf orce the effects 
enumerated under 1 through 4 above. 

In each experiment, over the range of aortic 
pressures and flows observed, the increase of ven- 
tricular stroke work was several times the simul- 
taneously observed increase in total peripheral re- 
sistance when carotid pressure was lowered. Thus 
rather than acting primarily to safeguard blood 
flow to the vital organs such as the brain and 
heart, the baroceptor acts not unlike a voltage 
regulating element which causes an increased in- 
put into an electronic system so as to maintain a 
constant voltage when the current requirements 
of the system it is supplying are increased. Thus 



68 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the carotid sinus helps to regulate the blood flow 
to all the tissues. 

A Comparison of the Hemodynamic Effects of 
Pacing the Atrium and Ventricle at the Same Rate 

By causing the atrium to contract while the 
atrioventricular valve is closed, the importance of 
the atrial contribution to ventricular filling was 
further shown. 

By closing the door, so to speak, on the atrium 
during atrial systole and thus depriving the ven- 
tricle of the filling pressure and fiber length it 
would otherwise have achieved, the ventricular 
end diastolic pressure was significantly lowered 
and the external work produced was thereby 
lessened. It was further observed that the amount 
of work produced by the ventricle from any given 
end diastolic pressure was lower during ventricu- 
lar pacing than during atrial pacing. Analysis of 
high speed ventricular pulse contours showed that 
the total ventricular effort is appreciably less con- 
certed and synchronous. Thus, when the first 
fibers are contracting, the flaccidity of those which 
are not as yet activated tends to impose the same 
hydraulic limitations as a ventricular aneurysm, 
e.g. diminish the effectiveness of the contraction. 
Similar considerations apply to the last contract- 
ing fibers. 

The Analysis of Coronary Sinus Blood for Cate- 
chol Amines Before, During and After Sympa- 
thetic Stimulation of the Heart 

The concentration of norepinephrine in coro- 
nary sinus blood rises sharply during cardiac sym- 
pathetic nerve stimulation. An interesting lead, 
however, is the massive outpouring, after the ces- 
sation of sympathetic stimulation, of a substance 
which assays chemically as norepinephrine by the 
Weil-Malherbe and Bone technique. This is prob- 
ably not norepinephrine since the heart's action 
does not conform to the presence of large amounts 
of the active substance at that time. 

The Influence of the Vigor of Atrial Systole on 
Closure of the Mitral Valve 

In the dog with heart block, between two and 
five atrial A-waves, and their reflections on ventric- 
ular diastolic pressure, can be studied in the ab- 
sence of the disturbances ordinarily produced by 
ventricular activity. As evidenced by the staircase 



pattern or its absence in the ventricular diastolic 
tracing, it can be determined whether the mitral 
valve has closed after any given atrial systole. 
The "nonclosing" atrial systoles are transformed 
by sympathetic stimulation into "closing" atrial 
systoles. The greater speed of the decline in 
atrial pressure is seen after the strong atrial A- 
wave causes the leaflets of the mitral valve to close, 
since the increased rate of change of atrial pres- 
sure after a strong atrial systole produces an ini- 
tially higher velocity of refluxing blood and thus 
is likely to close the valve. 

Auto-Regulation of the Performance Character- 
istics of the Ventricle 

The ventricle of the isolated heart, beating at a 
constant rate and free of reflex or hormonal regu- 
latory influences, requires no longer a time to put 
out any given stroke volume against a high aortic 
pressure than against a low one. If the work of 
the ventricle is increased solely by increasing 
aortic pressure, the ventricular function curve is 
much steeper than if the ventricular work is in- 
creased solely by increasing stroke volume. 
"When oxygen consumption is increased by in- 
creasing aortic pressure while holding stroke 
volume constant, there is a greater relative in- 
crease in coronary blood flow and a narrowing of 
the arteriovenous 2 difference. Conversely, 
when 2 consumption is increased by increasing 
stroke volume while holding mean aortic pressure 
constant, the increase in coronary blood flow is 
less marked and there is a widened arteriovenous 
2 difference. 

Reexamination of these phenomena showed 
that the effects of raising aortic pressure by in- 
creasing the resistance to left ventricular ejection 
produced alterations in the pulse contours which 
were in every respect similar to those observed 
after the administration of norepinephrine. 

There is a body of evidence suggesting that the 
responsiveness to catechol amine stimulation is a 
function of the biochemical environment of the 
stimulated effector organ. It is proposed that in- 
creased aortic pressure, by increasing coronary 
flow relative to 2 utilization, so alters the bio- 
chemical environment of the myocardium as to 
render it more responsive to catechol amines and 
thus increases the effective catechol amine stimu- 
lus. This hypothesis is to be investigated further. 



NATIONAL HEART INSTITUTE 



69 



STUDIES OF CIRCULATORY REGULATION- 
EXERCISE 

The total flow to both lower extremities was 
metered, before and during exercise, while the ar- 
teriovenous 2 difference, arterial and venous 
p0 2 and pH were recorded. Prior to exercise, 
three types of sympathetic stimulus were applied : 
(a) the reflex increase in sympathetic tone conse- 
quent to lowering carotid sinus pressure, (b) the 
emphatic sympathetic stimulation resulting from 
stimulating the central cut end of the vagus nerve, 
and (c) the injection of constricting doses of 
norepinephrine into the arterial line supplying 
the lower extremities. These were then repeated 
during simulated exercise induced by electrical 
stimulation of the muscles of both lower extremi- 
ties. In a second type of experiment the blood 
flow to each lower extremity was separately 
metered so that one extremity, the resting extrem- 
ity, could act as the control while the opposite 
extremity was exercised. 

The results of both types of experiments make 
it clear that with augmented muscular activity, 
the vascular bed of the active area can disregard a 
sympathetic stimulus to which it would ordinar- 
ily be responsive. This might be termed the func- 
tional sympathectomy of activity. The basis of 
this phenomenon is not yet known but it does not 
appear to be p0 2 . 

All of the studies in this laboratory have been 
greatly facilitated by the improvement of instru- 
mental and recording techniques, developments 
which have been largely carried out in this labo- 
ratory with some advice and assistance from the 
Laboratory of Technical Development. 

ABDOMINAL PRESSORECEPTORS 

It was earlier observed that splanchnic or pan- 
creatic vascular hypotension will produce tachy- 
cardia and a pressor response in the cat. In 
current experiments the lower abdominal aorta, 
near the bifurcation, is perfused at constant flow 
and the femoral arterial pressure recorded. The 
increased femoral artery pressure in the area per- 
fused at a constant flow, which takes place when 
the coeliac and superior mesenteric artery are oc- 
cluded, suggests that a reflex increase in periph- 
eral vascular resistance takes place. It remains, 



however, to exclude the possibility that the ob- 
served elevations of pressure in this vascular bed 
are not due to the influence of collateral channels. 

STUDIES OF THE CALLICREIN SYSTEM 

The objective of these investigations was to ob- 
tain an increased understanding of the physio- 
logical significance of this system for circulatory 
regulation. Callicrein, a hypotensive proteinase, 
acts on callidinogen, an alpha-2 globulin in plas- 
ma, to release a polypeptide called callidin which 
produces the observed vasodilatory effect. Since 
various callicreins are found in the urine, pan- 
creas, plasma, saliva, etc., and these callicreins 
may be differentiated by differences in their sus- 
ceptibility to proteolytic inhibitors, it is proposed 
that the callicreins are limited to local vasomotor 
regulation, the magnitude of the effect being de- 
termined by the activity of the tissue or organ 
involved. 

During the past year, semiquantitative methods 
have been developed for the biological determina- 
tion of some of the various components, e.g. calli- 
crein, callidinogen, callidin, and two plasma calli- 
crein inhibitors. The callidin inhibitor can be 
estimated by measuring the rate of destruction of 
callidin following its liberation from callidinogen. 
However, a quantitative method for the measure- 
ment of this inhibitor remains to be developed. 
With the techniques currently available, plasma 
and urine from patients with orthostatic hypoten- 
sion show a greater deviation from their normal 
controls than do plasma and urine from patients 
with essential hypertension. 

Plasmin, the proteolytic activity of serum found 
in the euglobulin fraction, has definitely been dis- 
tinguished from plasma callicrein. Other factors 
in plasma which have not yet been distinguished 
from plasma callicrein are the glass activated pro- 
teinase of Anderson and coworkers, Hageman's 
factor, and the permeability factor of Miles and 
Wilhelm. Studies of comparison of these factors 
have been initiated and no differences have yet 
been found. 

Because plasma callicrein was inhibited in vivo 
with diisopropylfluorophosphate, a known inhibi- 
tor of esterolytic activity, it was thought possible 
that the callicreins might be capable of digesting 
synthetic esters. Tosyl L-arginine methyl ester 



70 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



(TAME) has been found to be a substrate for the 
various callicreins. Crude urinary callicrein at- 
tacks this substrate at the same ratio of activity 
as does highly purified hog pancreatic callicrein 
(obtained from Dr. Moriya, Japan), and it is 
possible that urinary callicrein may be the main 
esterase excreted in the urine. The addition of 
acetone to human plasma, a procedure which 
causes the activation of plasma callicrein, activates 
at least two esterases; one inhibitable, the other 
not, with soy-bean trypsin inhibitor (SBI). To 
date, correlation between SBI inhibitable protein- 
ase and plasma callicrein has been consistent. It 
is possible that the SBI resistant proteinase may 
be the enzyme (callicreinase) responsible for the 
liberation of callicrein from its inactive precursor 
(callicreinogen). Plasma callicrein, when meas- 
ured as a proteinase inhibitable by SBI, appears 
to be eight times more active as a TAME esterase 
than is either urinary or pancreatic callicrein. 
Further studies will be required to determine 
whether acetone causes the activation of more 
than one proteinase inhibitable with SBI. 

REFLEXLY INDUCED CHANGES IN RENAL 
FUNCTION 

During stimulation of the isolated stellate 
ganglion of the anesthetized dog there is a diuresis 
which is not related to the changes in arterial 
blood pressure. The diuresis is characterized by 
a rapid onset, little or no change in urine solute 
or chloride concentration, an increase in glomeru- 
lar filtration rate and a rapid cessation with the 
"stoppage" of stimulation. Also, the diuresis 
occurs in the presence of a lowered left atrial pres- 
sure. It was further found that cervical vagotomy 
diminishes the extent of the diuresis whereas 
vagotomy at the level of the diaphragm does not 
appear to modify it. From the results of these 
experiments it was suggested that the increased 
urine flow during stimulation of the stellate gan- 
glion could be attributed to a peripheral vasodila- 
tation elicited via the baroreceptors in response to 
the changes in dynamic pressure effected by stel- 
late stimulation. Vagotomy effectively deprived 
the organism of a portion of the pressure-stabiliz- 
ing system, thus lessening the extent of the reflex 
changes induced by stellate stimulation and thus 
lessening the extent of the changes in renal vaso- 



motor tone, glomerular filtration rate, and urine 
flow. 



Laboratory of Kidney and Electrolyte 
Metabolism 

Five major areas of research are being pursued 
in the Laboratory of Kidney and Electrolyte 
Metabolism. These include: (1) studies on the 
mechanism of electrolyte excretion and acidifica- 
tion of the urine in intact animals; (2) studies on 
the mechanism of urinary dilution and concentra- 
tion; (3) studies of electrolyte and water trans- 
port in isolated systems, both living and artificial ; 
(4) studies of the control of aldosterone secretion 
in dogs; and (5) studies of a mammalian cardio- 
tonic protein system. 

Drs. Orloff and Burg have completed their ex- 
amination of the effect of the cardiac aglycone, 
strophanthidin, on electrolyte excretion in the 
chicken. As noted in the previous annual report 
they were able to elicit unilateral natruresis in the 
chicken by injecting strophanthidin into the renal 
portal venous system. Associated with the dimi- 
nution in Na + reabsorption they also observed 
interference with both K + and H + secretion. 
These effects have since been shown to be inhibited 
to a considerable degree by the simultaneous ad- 
ministration of K salts. The effects of strophan- 
thidin are unlike those observed following the in- 
jection of other pharmacological inhibitors of Na 
reabsorption. In the latter instance a fall in Na 
reabsorption is generally attended by reciprocal, 
rather than parallel changes in the secretion of K 
and H ions. On the basis of these data, as well as 
the results of studies of the kinetics of K + trans- 
port in renal cortical slices of rabbits, to be sum- 
marized below, it has been concluded that stro- 
phanthidin exerts its renal effect by inhibiting a 
contraluminal Na-K exchange pump. The latter 
pump is thought to be similar to that present in 
most living cells and presumably involved in the 
maintenance of the intracellular concentrations of 
Na and K. By interfering with the normal proc- 
esses of Na ejection and K uptake on the contra- 
luminal border, transluminal transport of these 
ions as well as of H + is depressed. 

The thesis that strophanthidin affects a hypo- 
thetical contraluminal exchange system derives 
further support from other studies of Drs. Orloff 



NATIONAL HEART INSTITUTE 



71 



and Burg. These workers reexamined the effect 
of various inhibitors, including strophanthidin, on 
K transport in rabbit renal cortical slices. The 
methods used were described in detail in the previ- 
ous report. They confirmed their earlier findings 
that strophanthidin reduces intracellular K and 
raises that of Na ; and that the effect is due in part 
at least to specific interference with K influx. 
These changes are consistent with an effect on 
linked Na-K exchange, analogous to that observed 
in red cells, muscle, etc. by other workers. 

Drs. Orloff and Burg were unable to demonstrate 
an antagonistic effect of adrenal steroids and 
strophanthidin on electrolyte transport as had 
been described by others. The effects of the agly- 
cone on electrolyte excretion are not altered by po- 
tent adrenal steroids (including aldosterone) ; the 
strophanthidin effects on K + and Na + accumulation 
in cortical slices of both normal and adrenalec- 
tomized animals are not altered by salt-active ster- 
oids ; nor is the inhibition of PAH accumulation 
by strophanthidin altered by aldosterone. 

Drs. Kahn, Brenes, Eaiiey, and Orloff are in- 
vestigating the effects of the infusion of ammo- 
nium salts on electrolyte excretion in the chicken. 
They have confirmed an earlier finding of this lab- 
oratory that the infusion of a series of ammonium 
salts into the leg veins elicits a profound natru- 
resis. The mechanism of this effect is being ac- 
tively studied. 

Drs. Jaenike and Berliner have concluded stud- 
ies utilizing a modification of the stop-flow tech- 
nique described originally by Malvin and Wilde. 
The technique involves occlusion of the ureteral 
outflow in the anesthetized dog for a variable pe- 
riod of time, release of the occlusion and collection 
of serial urine samples. The composition of these 
samples is thought to reflect to a considerable ex- 
tent the composition of the "stopped" intralumi- 
nal fluid at various levels in the nephron. The 
modifications introduced were designed to permit 
critical examination of distal tubular functions by 
effectively eliminating renal pelvic dead space 
(filling it with mineral oil) and isotopically 
labeling distal convolution fluid (subcapsular in- 
jection of K 42 during stop flow). The data con- 
firm that vasopressin markedly affects the perme- 
ability to H 2 of the distal convolution and 
further that electrolyte is also abstracted in this 
area. These results are consistent with a view of 



the mechanism of urinary dilution and concentra- 
tion previously described by Dr. Berliner and his 
associates. Utilizing this technique Drs. Jaenike 
and Berliner have also been able to reaffirm the 
view that K + and Na + are exchanged in the distal 
system and have clarified a number of factors in- 
fluencing this process. 

These investigators are at present studying the 
effect of vasopressin on the permeability of the 
collecting system to urea in the dog. The data 
support the interpretation that as vasopressin en- 
hances bulk flow of water along its osmotic gra- 
dient out of the collecting system into the inter - 
stitium, urea movement is accelerated in the same 
direction. This concept is of considerable signifi- 
cance with respect to the role of urea in the con- 
centrating mechanism. The medullary urea/ 
urine urea concentration ratio approaches unity in 
the presence of vasopressin at a time when flow of 
H 2 out of the collecting system into the medulla 
is high, and is considerably lower when no vaso- 
pressin is present and there is limited movement 
of water (and of urea) . 

Dr. Bray has investigated the osmotic pressure 
of rat kidney slices by a modification of a tech- 
nique which depends on the direct observation of 
the relative thawing time of previously frozen 
slices. The presence of a progressive increase in 
osmotic pressure from cortex to inner medulla in 
kidney slices of either dehydrated or non-dehy- 
drated rats has been confirmed. The cortex, 
which, for the most part, is isosmotic, contains 
some tubules which are definitely hypotonic. The 
hypotonic tubules are not seen in the outer medul- 
la. In contrast, the inner medulla which is 
considerably more concentrated, contains no de- 
tectably hypotonic structures. The collecting 
ducts in the inner medulla generally seem less 
concentrated than surrounding structures. In 
animals undergoing a water diuresis, the concen- 
tration gradient is less marked; the cortex re- 
sembles that of dehydrated animals as does the 
outer medulla. The inner medulla on the other 
hand is considerably less concentrated than in de- 
hydrated rats but the smaller structures (loops, 
capillaries) are definitely hypertonic and, as ex- 
pected, the collecting ducts are distinctly hypo- 
tonic. 

A clinical situation pertinent to the problem of 
urine concentration and dilution is being: studied 



72 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



by Drs. Earley and Orloff. Nephrogenic diabetes 
insipidus, a disease characterized by inability to 
elaborate a hypertonic urine due to insensitivity 
to vasopressin has been reported to respond to a 
limited degree to chlorothiazide, a diuretic agent. 
This observation has been confirmed and the 
mechanism of the drug's action is under study. 

Dr. Hoffman is continuing his investigation of 
the characteristics of electrolyte transport in 
human red cell ghosts (hemolyzed red cells) . He 
has established that the ghost system transports 
Na + in exchange for K + essentially as does the in- 
tact red blood cell. Thus electrolyte transport in 
ghosts has three components. (1) active trans- 
port; (2) passive transport, and (3) exchange dif- 
fusion. Active transport of Na out of cells re- 
quires the presence of K in the extracellular phase 
and is blocked by strophanthidin. Although this 
is similar to the situation in intact human cells, 
the kinetics of the process differs, and indicates, 
in contrast to the red cell findings, that a single Na 
compartment exists. 

In view of the precise definition of the ghost 
transport system, it has been possible to utilize it 
as an assay system in an attempt to define the 
metabolic intermediates involved in active trans- 
port. Dr. Hoffman has been able to show that 
electrolyte is immediately and completely de- 
pendent upon the availability of adenosine tri- 
phosphate; further, any reaction capable of gen- 
erating ATP stimulates the transport system as 
does ATP alone. It appears that the pump itself 
is, or has an an intimate component in its structure, 
ATPase. These observations together with re- 
lated findings in other laboratories constitute a 
major advance toward an understanding of the 
mechanism of active electrolyte transport. 

In association with Dr. Hoffman, Dr. Sidel and 
Dr. Kyan are exploring the possibility of measur- 
ing the rate of flow of H 2 across the red cell mem- 
brane under an osmotic gradient utilizing a rapid 
flow system developed by Dr. Tosteson, a former 
member of this laboratory. The method allows 
for rapid and continuous separation of extracel- 
lular fluid from a flowing system of suspended 
cells. They are also examining the effect of 
various liquid membranes (organic solvents) on 
the movement of Na and K from aqueous phases 
separated by the solvent membrane. They have 
established that the addition of cephalin to the 



membrane confers a slight degree of specificity to 
the movement of Na and K from one aqueous 
phase to the other. Other parameters are being 
studied. 

Dr. Cotlove, in association with Dr. Hogben 
(now at George Washington University), has ex- 
amined the kinetics of chloride transport across 
isolated frog gastric epithelium. They have 
established that chloride movement is more rapid 
across the luminal than the nutrient membrane 
of the epithelial cell. Using tracers and an inhibi- 
tor which affects only active transport and ex- 
change diffusion (DNP), they have concluded 
that the drug interferes with luminal transport 
only, presumably indicating this to be the site of 
linked, carrier-mediated, transport. Dr. Cotlove 
has also continued his examination of the so-called 
"true" chloride method, described in detail in the 
last report. 

Dr. Davis and his associates have extended their 
studies of the mechanism of aldosterone secretion 
in dogs and have restudied the pituitary role in 
this process. Although administration of a low 
salt diet or constriction of the thoracic inferior 
vena cava stimulates hormone secretion in normal 
animals, no such effect was observed following 
hypophysectomy. Administration of ACTH re- 
stores the capacity to respond to vena cava con- 
striction with an increase in the aldosterone out- 
put in adrenal vein blood. That this effect of 
ACTH is permissive is indicated by studies in 
unanesthetized dogs. The stress of anesthesia and 
operation induces ACTH secretion and a high 
corticosterone output in the usual anesthetized 
animal. In the unanesthetized trained dog corti- 
costerone output is low, indicating that ACTH 
secretion is not increased. Nevertheless caval con- 
striction stimulates aldosterone output just as in 
anesthetized animals, without an increase in cor- 
ticosterone secretion. It is probable that ACTH 
secretion is normal in the secondary hyperaldo- 
steronism of caval constricted animals, and that 
ACTH merely serves to support aldosterone pro- 
duction at a high level rather than initiate its 
secretion. 

Attempts to characterize the precise mode of 
stimulation of aldosterone secretion are continu- 
ing. It is now apparent on the basis of work re- 
ported in the past by this group that an unknown 
trophic hormone is involved in the increased se- 



NATIONAL HEART INSTITUTE 



73 



cretion of aldosterone in animals with constric- 
tions of the inverior vena cava. Efforts to isolate 
and identify this hormone are being carried out 
in collaboration with Dr. Titus. 

Chronic denervation of the cervical common 
carotid, the carotid sinus, and the cervical por- 
tions of the external and internal carotid had no 
significant effect on either electrolyte balance or 
aldosterone excretion in normal dogs or dogs with 
constrictions of the vena cava. However, in one 
dog aortic denervation subsequent to caval con- 
striction and cervical carotid denervation dimin- 
ished aldosterone excretion without affecting so- 
dium balance. Furthermore, in one other dog, 
constriction of the abdominal arteries (coeliac, 
superior, and inferior mesenteries) increased ar- 
terial pressure and aldosterone secretion mark- 
edly. Although as yet inconclusive, these data 
are concordant with the possibility of neural re- 
ceptors somewhere in the vascular tree. Studies 
of this nature are being pursued. Neither mid- 
brain transection nor pinealectomy has been 
shown to appreciably affect aldosterone secretion 
and animals with such lesions appear to respond 
normally to stimuli known to enhance aldosterone 
output in unoperated controls. 

Drs. Hajdu and Leonard are continuing their 
study of a mammalian cardiotonic protein sys- 
tem, discovered and characterized by them in the 
past. Recent efforts have been devoted to the as- 
sessment of the physiological significance of this 
system in man and animals. The concentration 
of the cardiotonic system is distinctly elevated in 
a small group of patients with uncomplicated hy- 
pertension and in patients with aortic stenosis. 
It is markedly diminished in a considerable pro- 
portion of patients suffering from congestive 
heart failure of unknown origin (that is not due 
to the usual causes, such as hypertension, rheu- 
matic heart disease, etc.). The activity virtually 
disappeared from the plasma of patients sub- 
jected to extracorporeal circulation and returned 
to normal in all but one who developed irreversi- 
ble vascular collapse. These preliminary data 
warrant the tentative hypothesis that the system 
has specific cardiotonic function in man, the ac- 
tivity increasing in a compensatory fashion in 
disease states associated with increased isometric 
tension of the left ventricle, and being diminished 
in severe myocardial insufficiency. Animal stud- 



ies are in progress in an attempt to delineate the 
function of the system in a more precise manner. 

Laboratory of Clinical Biochemistry 

The problem of amine formation and metabo- 
lism has been reinvestigated to determine the 
nature of the decarboxylation of the various amino 
acids and the routes of metabolism of the resulting 
amines. By the use of column and paper chro- 
matography it was possible to demonstrate that 
human urine from normal individuals (particu- 
larly following administration of monoamine 
oxidase inhibitors) contains at least 25-30 amines. 
In addition to those already known, it was possi- 
ble to identify ortho-tyramine, meta-tyramine, 
and phenylethylamine. The presence of so many 
amines suggested either that there must be many 
individual amino acid decarboxylases or that the 
available enzymes were not very specific. The 
enzyme known as 5 -hydroxy tryptophan (5HTP) 
decarboxylase, when purified, was found to de- 
carboxylate DOPA, 5HTP, tryptophan, phenyl- 
alanine, tyrosine and histidine, at rates in the 
order listed. Further work is proceeding on the 
assumption that a single enzyme, analogous to L- 
amino acid oxidase in its lack of specificity, is re- 
sponsible for catalyzing all these reactions. This 
finding is of great significance from a theoretical 
as well as a practical standpoint. First, it means 
that a large number of dietary and metabolically 
formed amino acids continually give rise to a 
spectrum of amines. The amount of a given amine 
which appears in the tissues is then dependent 
upon the affinity of the decarboxylase for the pre- 
cursor amino acid, the concentration of the amino 
acid in the tissues, the presence of competing 
amino acids, and finally on its rate of destruction 
by metabolizing enzymes. The latter two factors 
may vary significantly in many pathologic condi- 
ditions. As for amine metabolism, it is now ap- 
parent that a variety of mechanisms are available. 
Although all of the aromatic and cyclic amines 
are metabolized, to some extent, by monoamine 
oxidase (MAO), there are more specific enzymes 
available for the metabolism of some of the 
amines. Thus histamine may be metabolized by 
diamine oxidase as well as an N-methyl pherase. 
In the case of the epinephrines, it appears that 
once they are introduced into the circulation, 
methylation is the major route of metabolism. 



74 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



However, it would appear from studies of Dr. 
Udenfriend and his associates that metabolism of 
norepinephrine in brain, heart and other organs 
is largely due to MAO. The complexity of the 
effects produced by inhibitors of amine metabo- 
lism is no longer surprising when account is taken 
of the fact that serotonin and norepinephrine are 
but two of the many amines affected. The 
marked central effects of tryptophan and trypt- 
amine on patients receiving MAO inhibitors com- 
prise but one example of the significance of the 
totality of amines. 

In view of all these factors an investigation of 
the biochemical effects of inhibitors of amino acid 
decarboxylation in patients was undertaken in col- 
laboration with the Section of Experimental Ther- 
apeutics, a Methyl DOPA was found most effec- 
tive in this respect and following its administra- 
tion tyramine formation was found markedly di- 
minished; the excretion of tryptamine and sero- 
tonin was also decreased and it appears that there 
may have been some detectable effects on norepine- 
phrine formation. Simultaneous studies with 
purified mammalian decarboxylase indicate that 
a-methyl DOPA inhibits formation of all aro- 
matic and cyclic amines including histamine. The 
finding that a-methjd DOPA is a potent antihy- 
pertensive agent (see report from Section of Ex- 
perimental Therapeutics) is most interesting and 
gratifying. However, the biochemical findings in- 
dicate that the mechanism of its action is not yet 
clear and it does not appear to be attributable en- 
tirely to diminished formation of norepinephrine. 

In view of these interesting and important find- 
ings, studies of a number of additional aspects of 
amine metabolism have been undertaken. Con- 
version of tryptamine to 6-hydroxytryptamine 
was shown to be catalyzed by liver microsomes 
and TPNH. It may be that ortho-tyramine may 
be formed from phenylethylamine in a similar 
manner. The mechanism of formation of meta- 
tyramine is not yet clear but may involve interme- 
diate formation of meta-tyrosine through the ac- 
tion of phenylalanine hydroxylase. Methods have 
been developed for the determination of trypta- 
mine and tyramine in tissues and in urine. Stud- 
ies with kynuramine, the amine derived from 
kynurenine, have led to a simple and direct spec- 
trophotometric procedure for assaying MAO. 



The aldehyde formed from kynuramine cyclizes 
so readily to 4-hyclroxyquinoline that in the crud- 
est preparations spectrophotometry can be used as 
a rapid and direct assay for oxidative deamina- 
tion. This method may be expected to facilitate 
markedly steps leading to purification and charac- 
terization of MAO. 

In studies of catecholamine metabolism the en- 
zyme dopamine /^-oxidase was shown to be pres- 
ent in as high concentration in hypothalamus and 
caudate nucleus as in adrenal medulla. However, 
little if any was found in the higher centers of 
the brain. The mechanism of the dopamine /?-oxi- 
dase catalyzed reaction has been under investiga- 
tion in collaboration with Dr. Witkop's labora- 
tory. One of the results of these studies was the 
demonstration that dopamine gives rise to 2,4,5- 
trihydroxyphenylethylamine upon chemical oxida- 
tion and that this product appears in the urine 
when dopamine-C 14 is administered to animals 
and patients with pheochromocytoma. 

Studies of the mechanism of serotonin uptake 
by platelets have continued. Using saline media 
it has been possible to obtain additional evidence 
that this process is one of active transport. Re- 
quirements for K + and PO = were demonstrated, 
the demonstration of inhibition by digitoxin being 
further evidence of a K + requirement. A relation- 
ship between serotonin uptake and glycolysis was 
also shown, including marked inhibition by 
fluoride. 

Mechanisms whereby amino acids penetrate into 
various mammalian cells are also under study. 
It has been possible to show that L -tyrosine is 
taken up from blood into brain in vivo by a proc- 
ess of facilitated transport. The evidence is that 
the Z-isomer penetrates rapidly and several times 
faster than the Z>-isomer and more rapidly than 
non-amino acid congeners. The uptake of L- 
tyrosine is markedly inhibited by other aromatic 
amino acids including tryptophan and fluorophen- 
ylalanine but is not inhibited by alanine, histidine 
or lysine. Although tyrosine is rapidly taken up 
into muscle too, this process is not inhibited by 
other amino acids. Using rat diaphragm muscle 
it has thus far not been possible to detect any 
evidence of active or facilitated transport of tyro- 
sine, the amino acid entering by diffusion only. 
These findings are contrary to conclusions put for- 



NATIONAL HEART INSTITUTE 



75 



ward by Christiansen and others. The studies 
will be extended to other tissues and to other 
amino acids. 

There have been some interesting findings relat- 
ing to y-aminobutyric acid. Transamidination 
has been shown to occur in brain and to yield y- 
guanidinobutyric acid. A peptide containing y- 
aminobutyric acid and histidine and possibly an- 
other amino acid has been found in brain. In 
beef brain it is present in amounts as high as sev- 
eral milligrams per cent. Histidyl-y-aminobu- 
tyric acid has already been synthesized by Dr. L. 
Cohen of Dr. Witkop's laboratory to help in the 
investigation of structure. 

Studies on the metabolism of amino acids unique 
to collagen have been continued. It was shown 
that ascorbic acid which influences collagen forma- 
tion does not do so by influencing hydroxyproline 
formation. Ketoproline increases hydroxyproline 
levels in tissues. Investigation of this phenom- 
enon showed that ketoproline inhibits hydroxy- 
proline metabolism by liver and by bacteria, and 
is itself converted to hydroxyproline. A mam- 
malian enzyme system which catalyzes this conver- 
sion in the presence of DPN was studied and 
found to be distinct from other dehydrogenases. 
Studies in this laboratory have corroborated the 
reported presence of ketoproline in actinomycin 
and have shown that ketoproline is of the L con- 
figuration. The metabolism of another amino acid 
found only in collagen was also investigated. Hy- 
droxylysine was found to be metabolized by achro- 
mobacter and liver microsomes to 5 -hydroxy - 
pipecolic acid and l-amino-5-hydroxyadipic acid. 
A sensitive and specific method for measuring hy- 
droxyproline in tissues was developed. It has 
been applied to studies on urinary hydroxyproline 
in man and animals. Using proline-C 14 it was 
shown that in rats urinary hydroxyproline be- 
comes labelled. The rate of disappearance of C 14 
from urinary hydroxyproline in adult rats indi- 
cates three components. The first may represent 
a rapid conversion of proline to free hydroxy- 
proline ; the second represents a hitherto unknown 
peptide material with a fairly rapid turnover 
(half life ca 15 days) ; the third is typical of the 
slow turnover of collagen hydroxyproline which is 
known to obtain in adult animals. 



SECTION ON EXPERIMENTAL THERA- 
PEUTICS 

The principal areas of investigation in this sec- 
tion may be grouped for descriptive purposes as 
(1) studies on vasoactive substances, (2) metabo- 
lism of amino acids in man, and (3) action and 
metabolism of drugs. 

Vasoactive Substances 

Accumulated experience in this laboratory on 
the differential assay of urinary catecholamines in 
pheochromocytoma (18 cases) plus a review of 
such assays reported in the literature (60 cases) 
indicates the procedure has usefulness in locali- 
zation as well as diagnosis of the tumor. With 
exception of two tumors of the Organs of Zucker- 
kandl, excessive excretion of epinephrine rep- 
resented a tumor in the adrenal area. In collabo- 
ration with Dr. J. Pisano (LCB) methods were 
developed for measuring the m-O-methyl metabo- 
lites of epinephrine and norepinephrine. In 
studies on the urine of 15 patients with pheo- 
chromocytoma, the free catecholamines accounted 
for 0.4-6.7 percent, the methoxy-catecholamines 
for 17-42 percent and 3-methoxy-4-hydroxy- 
mandelic acid for 57-78 percent of the total ex- 
cretion of catecholamines plus metabolites. It is 
felt that catecholamine assays may be supplanted 
by measurements of metabolites for the initial 
chemical detection of pheochromocytoma. 

A method for estimating catechol-O-methyl- 
pherase activity in man has been developed. It 
consists of administering the d-isomer of isopro- 
terenol and determining the percentage of the 
dose excreted in the urine as the O-methyl metab- 
olite. Use of this technique along with methods 
for measuring monoamine oxidase (MAO) activ- 
ity in vivo have revealed no differences between 
normal subjects and those with primary hyper- 
tension. This suggests that if an abnormality in 
the metabolism of norepinephrine in hypertension 
exists, the defect lies in biogenesis of the amine or 
in some other mechanism for inactivation such as 
protein -binding. 

The further study of amines excreted in the 
urine of patients receiving MAO inhibitors has 
led to conclusive identification of m-tyramine and 



76 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



phenylethylamine. In two cases of phenylketo- 
nuria the rise in urinary phenylethylamine upon 
the administration of MAO inhibitors was con- 
siderably greater than normal while the rise in 
m-tyramine excretion was subnormal. The first 
finding represents the only demonstration of ex- 
cess amounts of a centrally-active compound in 
phenylketonuria and the latter suggests that the 
defective hydroxylation of phenylalanine in this 
condition may involve the meta as well as the para 
position. 

The cardiovascular actions of various amines 
are being investigated systematically in dogs and 
in patients. Many different amines increase con- 
tractile force and potentiation of the effect of 
these amines has been observed during MAO in- 
hibition in the case of phenylalkylamines which 
lack a B-hydroxyl group and/or alkyl substitution 
on the amino group. These factors also deter- 
mine susceptibility as substracts for MAO. Most 
attention has been given to norepinephrine, sero- 
tonin, dopamine, tryptamine and tyramine. 
Curiously, the administration of Ritalin produced 
an effect opposite to that of MAO inhibitors, with 
potentiation of the cardiac effects of norepineph- 
rine and serotonin but not of dopamine, trypt- 
amine and tyramine. In hypertensive subjects, 
potentiation of pressor responses to dopamine by 
MAO inhibitors was closely related to the degree 
of enzyme inhibition (as measured by urinary 
tryptamine) whereas potentiation of the pressor 
effects of norepinephrine and methoxamine oc- 
curred only when there was also sympathetic 
blockade as manifested by orthostatic hypotension. 

In cooperation with the Clinic of Surgery, ex- 
tensive studies of cardiac contractile force re- 
sponses in man have been done using the strain 
gauge arch technique. The findings were similar 
to those in the dog and thus resolve such old argu- 
ments between pharmacologists and clinicians as 
to whether norepinephrine is a cardiac stimulant 
in man and whether cardiac glycosides increase 
the contractile force of the "normal" human heart. 
The answer to each question is yes. 

Metabolism of Amino Acids in Man 

The method of assay for hydroxyproline 
(OPE.) in tissues and urine has been simplified 
considerably so that it is now possible to measure 
OPR routinely. A more extensive survey of con- 



nective tissue disorders has begun and arrange- 
ments have been made to perform a broader study 
of Marfan's Syndrome in 48 affected families 
under the care of Dr. V. McKusick of Johns Hop- 
kins Hospital. Study of the specific activity of 
urinary OPR in rats after injection of C-14 pro- 
line has given a measure of the turnover rate of 
OPR and presumably also of body collagen. Sim- 
ilar studies in patients appear feasible. 

Recently, the compound a-methyl-dihydroxy- 
phenylalanine (aM-DOPA) has become available 
for clinical studies. This substance has been found 
in LCB and elsewhere to be an effective inhibitor 
of various amino acid decarboxylases in vitro and 
in vivo in laboratory animals. Administration of 
the compound (2.0 gm./day) to human hyper- 
tensives has been shown also to produce decar- 
boxylase inhibition as indicated by a decrease in 
the excretion of tryptamine and tyramine follow- 
ing standard loading doses of tryptophan and 
tyrosine respectively. In the course of this work, 
a hypotensive response was also observed (see next 
section). aM-DOPA is only the first of several 
decarboxylase inhibitors to be studied and thus 
attempts are being made to develop procedures 
for use as indices of decarboxylation. Patients 
with pheochromocytoma, carcinoid syndrome, 
phenylketonuria and urticaria pigmentosa may 
prove helpful in these studies because of the ex- 
aggerated formation of amines in these conditions. 
By the same token, specific decarboxylase in- 
hibitors may be useful therapeutic agents in these 
disorders. 

Action and Metabolism of Drugs 

Five different MAO inhibitors have been evalu- 
ated for biochemical and pharmacologic activities 
in human hypertensives: iproniazid (Marsilid), 
l-phenyl-2-hydrazinopropane (JB-516, Catron), 
dl-phenylcyclopropylamine (SKF-385), nialam- 
ide (Niamid) and phenylethylhydrazine (Nardil). 
Although administration of each of these 
agents produced postural hypotension, a precise 
correlation could not be made between the 
degree of enzyme inhibition and hypotension. 
However, the dose of each drug required to pro- 
duce an increase of urinary tryptamine to levels 
of 500-700 /xgm./day was of a magnitude similar 
to that reported to give optimal psychiatric ef- 
fects. JB-516 is still the most potent and con- 



NATIONAL HEART INSTITUTE 



77 



sistently effective hypotensive agent to be found 
among this group of drugs. Studies at George 
Washington University Hospital on 30 patients 
over a period of 6 months confirmed its effective- 
ness particularly when used in combination with 
chlorothiazide. However, the development of 
visual toxicity (diminished acuity as well as color 
perception) which has been only slowly reversible 
in three cases seriously limits the use of this agent 
in the management of hypertension. The daily 
dose which may be administered safely is less than 
12 mg./day in our opinion and this amount is 
insufficient for control of the blood pressure in 
many hypertensives. The d and 1 isomers of 
JB-516 have been found to be equally effective for 
reducing the blood pressure but it is not yet known 
whether one of them will be devoid of visual 
toxicity. Complete absorption of JB-516 has 
been shown through the unusual experiment of 
demonstrating equivalent effects on urinary tryp- 
tamine excretion by single oral and intravenous 
doses of the drug. 

Studies of the actions of MAO inhibitors on 
the autonomic nervous system in dogs have shown 
sympathetic (but not parasympathetic) gangli- 
onic blockade with harmine and iproniazid but 
not with several other inhibitors. 

The finding that aM-DOPA produces lowering 
of the blood pressure in patients with hyperten- 
sion is under active investigation. A uniform and 
significant decrease hi both supine and standing 
blood pressure has been observed in several cases 
during short-term studies. 

Since June 1959 studies of anti-fibrinolytic 
agents have been conducted in collaboration with 
Professor J. Waldenstrom and associates of 
Malmo, Sweden. Several aliphatic amino acids 
were found to be inhibitors of plasminogen acti- 
vation in vitro, the most potent being A-amino- 
valeric acid, A-aminolevulinic acid and E-amino- 
caproic acid (E-ACA). Administration of the 
latter compound to two patients with pathologic 
fibrinolysis (secondary to leukemia and cirrhosis) 
has shown it to be effective in man. A method 
for chemical assay of E-ACA in urine was de- 
veloped and about 60 percent of a single dose (6 
gm.) given intravenously or orally to patients 
was found to be excreted within 12 hours. The 
hypothesis that the early stages of atherosclerosis 
are related to deficient fibrinolysis is an attractive 



one and for this reason a continued interest in 
synthetic and naturally occurring inhibitory sub- 
stances is contemplated. 

SECTION ON CARDIODYNAMICS 

It is the ultimate objective of this section to 
study the physiological behavior of the cardio- 
pulmonary system of human subjects as they go 
about their usual daily activities and as they are 
subjected to various physiologic, psychic, pharma- 
cologic, and other stressful interventions, both in 
health and disease. The measurement of a large 
number of physiologic variables under conditions 
most nearly simulating normal activity has made 
the development of new instrumentation methods 
of prime importance. Further advances of this 
broad approach will depend largely upon develop- 
ment of (1) new highly specialized instrumen- 
tation and (2) some new sophisticated biophysical 
and physiologic approaches. The activities of the 
Section have been determined largely by these 
needs. 

The accurate measurement of pressures has re- 
mained a problem of high priority in the Section 
since new approaches developed in this laboratory 
both in the vascular and the pulmonary field have 
placed ever increasing demands on the accuracy 
of the measurements. A paper reviewing pro- 
gress in this field will appear shortly. This sec- 
tion has worked with the Laboratory of Technical 
Development to develop an electrical pressure cor- 
rection device which will instantaneously and con- 
tinuously correct dynamic response errors in 
various pressure manometer systems. 

A major advance in the field of blood-flow 
measurement was achieved when the catheter- 
computed pressure gradient method for the in- 
stantaneous and continuous estimation of aortic 
blood velocity was developed for use in the intact 
man. This advance has opened the way to measur- 
ing the power output of the heart from moment 
to moment, the kinematics of cardiac ejection, the 
power loss at diseased valves, and the distributed 
impedances and junctional admittances in the 
vascular tree. Measurements of this kind will be 
necessary to evaluate the abnormal physical prop- 
erties and energetics of the vascular system in 
myocardial disease, coronary attenuation, arterio- 
sclerosis, hypertension and related conditions. To 



78 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



date, 19 studies, using the catheter-computer 
method for instantaneous blood velocity, have 
been performed without major complication and 
the data are undergoing analysis. The resources 
of the mathematical and computer section of NIH 
have been called upon for processing some of these 
data. 

Various electrical analogs of the vascular sys- 
tem are being devised and tested with the use of a 
Donner Analog Computer. The relationship be- 
tween the vascular visco-elastic properties and the 
transmitted pressure and flow wave are being 
studied in animals. Preliminary results indicate 
among other things that under certain circum- 
stances it should be possible to infer the character 
of the central pulse from measurement of periph- 
eral pressure pulses. The significance of this is 
two-fold: (1) Pressure pulse data may be useful 
in indirect determination of the visco-elastic 
properties of intact human vascular systems by 
the use of a relatively simple computer unit. (2) 
It may be possible to compute the instantaneous 
blood flow from the heart using peripheral pulse 
information. 

The implication of this latter possibility is im- 
portant in that it may make it possible to measure 
cardio-circulatory function under circumstances 
close to those of normal activity. The develop- 
ment of miniaturized transducers, amplifiers, te- 
lemetering systems and tape recording will be nec- 
essary for the ultimate realization of this goal. To 
this end pilot studies using a tape recorder have 
been started to investigate the feasibility of multi- 
channel tape recording in the physiologic applica- 
tion. Progress to date has suggested that many 
improvements must be made; however, the ap- 
proach appears entirely practical and the acquisi- 
tion of an improved system of this type will be 
necessary for the continuation of this work. 

Pilot studies in animals, using the blood veloc- 
ity catheter, are in progress to determine the ef- 
fect of impairment of the coronary circulation. 
Acute attenuation of the coronary circulation 
either by ligature or embolization of the coronary 
tree produces dramatic and reproducible changes 
in the blood velocity curve. Methods are being 
developed for producing chronic coronary insuf- 
ficiency in dogs. Pilot studies in dogs are in 
progress to determine the pressure-diameter rela- 
tionships in various parts of the systemic and 



pulmonary vascular bed so that inferences regard- 
ing flow can be made from the blood velocity 
curve. When these studies are completed, the 
problem of systemic and pulmonary vascular im- 
pedance can be better evaluated. Preliminary 
data indicate that although there is a marked di- 
vergence between the shape of the pressure and 
velocity curves in the aorta, their shapes become 
almost identical as either the systemic or pul- 
monary capillary bed is approached. This indi- 
cates that the arteriolar-capillary-venular bed 
probably behaves as a pure resistance without 
significant reactance. 

Vascular resistance in the pulmonary bed is be- 
ing studied both by the catheter blood velocity 
technique and by conventional dye dilution curve 
methods to establish comparison of the methods 
and to establish the effect of intrathoracic pres- 
sure and various pharmacologic agents on resist- 
ance. These studies are being carried out both in 
man and in animals. A new method was de- 
veloped for simplifying and making more re- 
producible the calibration of dye dilution curves. 

Since the direct measurement of the intratho- 
racic pressure in man is hazardous and in itself 
alters the normal function of the lung, studies are 
in progress to establish the relationship of in- 
trathoracic pressure to intraesophageal pressure. 
The determination of intrathoracic pressure is nec- 
essary for determination of the transmural stress 
on the intrathoracic structures in most of the fore- 
going studies. 

New methods of studying the mechanical be- 
havior of the lung were developed. Detailed 
studies of the unified pressure-flow-volume-time 
relationship of the living human lung were car- 
ried out in normal, cardiac and emphysematous 
subjects. A relationship between the maximum 
achievable expiratory flow and degree of lung in- 
flation was discovered which theoretically has far- 
reaching physical and physiological implications. 

Studies are being undertaken jointly with the 
Section on Experimental Therapeutics to assess 
the changes in flow and resistance occurring with 
the administration of inotropic and vaso-active 
agents in normal controls and patients with vari- 
ous disease states. The new approach to indicator 
dilution curves makes it possible to do a sizeable 
number of these determinations with markedly re- 
duced amount of time and effort. 



NATIONAL HEART INSTITUTE 



79 



Other studies are being undertaken to determine 
the incidence and character of arrhythmias in pa- 
tients after closure of atrial septal defects. These 
are done as a joint project with the Clinic of 
Surgery, NHI. Study of the characteristics and 
possible mechanisms of formation of ectopic beats 
during right and left heart catheterization is being 
pursued by members of the Section in association 
with Dr. Albert Kistin of the Beckley Memorial 
Hospital, Beckley, W. Va. This includes devel- 
opment of better intraesophageal and intracardiac 
electrodes. 

Studies of coronary flow dynamics in human 
patients are to be undertaken in an attempt to 
develop methods that are valid and clinically 
more useful. The analysis of nitrous oxide gas 
by newer techniques including gas chromatog- 
raphy has been explored and has promise. It 
also appears worthwhile to try to assess the possi- 
ble use of isotope tracer substances with external 
counting as a means for finding some index of 
myocardial blood flow. 

SECTION ON CLINICAL ENDOCRINOLOGY 

The activities of the Section on Clinical Endo- 
crinology may be grouped into four general areas, 
as follows : (1) studies on the function and metab- 
olism of steroids and their role in disease states; 
(2) studies of the abnormalities in water metabo- 
lism found in various disease states ; (3) studies of 
calcium metabolism, with special reference to the 
effects of parathyroid hormone, the effects of vita- 
min D, and the effects of calcium on renal func- 
tion; and (4) studies of the permeability of ar- 
teries to large molecules. 

Steroid Metabolism 

Studies in the area of steroid metabolism in- 
clude further exploration of the control of aldos- 
terone secretion, clinical studies in hyperadrenal 
corticism, and measurement of relation of steroid 
function to structure. 

Afferent pathways mediating control of aldos- 
terone secretion were explored. The decreased al- 
dosterone secretion which occurs upon release of 
construction of the inferior vena cava had been 
found to require the presence of the vagus nerves. 
It is likely that they arise in the area of the auricles 
and the great vessels. Studies were carried out to 



define the pathways required for the increased 
secretion of aldosterone which occurs upon appli- 
cation of caval construction. It was found that 
constriction of the carotid arteries was also an ef- 
fective stimulus to increased secretion of aldos- 
terone. Exploration of the carotid arteries in the 
dog revealed the presence of an area with slight 
baroceptor function in the region of the thyro- 
carotid arterial junction. This baroceptor func- 
tion could be abolished by denervation of this area. 
It was found that denervation in this area also 
abolished the rise of aldosterone secretion follow- 
ing constriction of the carotid arteries as well as 
that which follows constriction of the inferior 
vena cava. Analysis of the results suggests that an 
important stimulus to increase of aldosterone se- 
cretion depends upon arterial pulse pressure, and 
that the intracarotid pulse pressure is the most im- 
portant variable. 

In patients with aldosteronism, direct measure- 
ments of blood volume, pulse pressure, and potas- 
sium balance have been carried out. Extracellular 
fluid volume was changed by loading subjects with 
sodium or depleting them of sodium. Changes in 
intravascular volume were produced by the infu- 
sion of albumin. Finally, the action of aldo- 
sterone on the renal tubules was blocked with the 
use of aldosterone antagonists. With these meas- 
urements, it was hoped that primary aldosteron- 
ism, with autonomous secretion from a tumor, or 
unexplained "primary" hypersecretion from hy- 
perplastic glands, could be distinguished from sec- 
ondary aldosteronism. (Numerous studies, pre- 
viously reported, support the view that changes 
in intravascular volume have a major role in the 
control of aldosterone secretion in man, as in the 
dog with experimentally produced aldosteron- 
ism.) It was found that measures which changed 
intravascular volume would induce changes in al- 
dosterone secretion in patients with secondary al- 
dosteronism, but not in patients with primary al- 
dosteronism. The effect of aldosterone-blocking 
agents was more complex. Whereas in most cases 
of primary aldosteronism, secretion of aldosterone 
did not rise when the effect of the hormone was 
blocked, exceptions were seen when large increases 
in serum and total body potassium followed the 
use of the blocking agents. Under these circum- 
stances, aldosterone secretion might rise in pri- 



80 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



mary aldosteronism, as it regularly did in secon- 
dary aldosteronism. 

It was found that potassium deprivation would 
lower aldosterone secretion, and restoration of the 
deficit would elevate aldosterone secretion, as pre- 
viously reported. In carefully controlled balance 
studies, it was shown that this phenomenon could 
be produced without changes in the blood volume, 
as measured directly with double isotope dilution 
methods. 

Two patients with potassium-losing renal dis- 
ease were studied in an attempt to elucidate the 
relationship between aldosterone secretion and po- 
tassium loss. It was found that aldosterone secre- 
tion would change markedly in response to 
changes in sodium intake and, thus, could not be 
considered "primary" or "autonomous." The de- 
gree of potassium loss appeared to depend, in 
turn, upon the secretion of aldosterone. One sub- 
ject was explored and found to have hyperplastic 
adrenal cortices. The potassium loss was greatly 
improved following subtotal resection. 

Studies of aldosterone secretion in patients with 
postural hypotension have been continued. In 
view of the evidence that arterial pressure has an 
important role in the control of secretion, it was 
considered worthwhile to measure the efficiency of 
the control of secretion in as many subjects with 
this disorder as possible. Subjects were classified 
according to the location of lesion, using a series 
of tests, including mental arithmetic (which may 
reveal a normal efferent system and locate the es- 
sential lesion in afferent pathways) and periph- 
eral nerve block or vasoconstrictor agents (which 
may reveal an inactive, hypersensitive efferent sys- 
tem and locate the lesion in efferent pathways) . It 
was found that, whereas patients with afferent le- 
sions may show an inability to secrete aldosterone 
with sodium depletion, patients with efferent le- 
sions usually retain this property. 

Patients with Cushing's syndrome have been 
studied for relative dependence of hydrocortisone 
secretion upon blood levels of hydrocortisone or 
hydrocortisone analogs. These studies were done 
to clarify the locus of the essential lesion in Cush- 
ing's syndrome. All patients with hypersecretion 
of hydrocortisone were tested first with moderate 
doses of hydrocortisone analog. Under these con- 
ditions patients with Cushing's syndrome were 
found to excrete tetrahydrocortisone in unaltered 



quantities. They could be distinguished clearly 
from patients with ovarian disorder who may, 
at times, show hypersecretion of hydrocortisone, 
but a ready fall of secretion with the suppressive 
steroid. The patients with Cushing's syndrome 
were then subjected to suppressive doses four times 
as large. The response of the urinary steroids to 
this procedure allowed the separation of the 
patients into two groups: patients whose hydro- 
cortisone secretion was not suppressed had adrenal 
cortical adenomas ; with one exception, those who 
did show suppression had hyperplasia of the 
adrenal cortex. Subjects with Cushing's syn- 
drome are being further studied by measuring the 
response to agents which block the 11-hydroxyla- 
tion of steroids. It is hoped, in this way, to dis- 
tinguish Cushing's syndrome of pituitary origin 
from that of hypothalamic origin. 

Methodology has been developed for fractiona- 
tion of 17-ketosteroids, and patterns are being de- 
termined in patients with Cushing's syndrome, 
patients with the adrenogenital syndrome and 
patients with ovarian disease. In this way, it is 
hoped that more can be learned about the specific 
enzymatic defect in the various disorders. In 
particular, patients with the adrenogenital syn- 
drome may be classified in this way, as regards the 
presence or absence of 11-hydroxylase. 

The factors influencing the protein-binding of 
steroids in vivo have been further studied. The 
amount of hydrocortisone bound could be greatly 
increased by administration of estrogen. As this 
does not alter the metabolic effects attributable to 
hydrocortisone, it appears probable that the meta- 
bolic activity of circulating hydrocortisone de- 
pends solely upon the "free" fraction. A number 
of steroids were tested for their ability to bind to 
serum proteins. Studies of the effect of fasting 
and of surgical trauma on the binding of steroids 
are now in progress. 

Studies of the relation of steroid structure to 
steroid function have been continued, both in meta- 
bolic balance studies in man and in acute studies 
of renal sodium and potassium excretion in the 
adrenalectomized dog. It is hoped, with such 
studies, to achieve an understanding of the essen- 
tial features in steroid structure responsible for 
the various metabolic effects, and also to allow pre- 
diction of structural changes which might enhance 
the activities of steroids. 



NATIONAL HEART INSTITUTE 



81 



Studies of Disturbed Water Metabolism 

Studies have been carried out to define the ab- 
normality in water metabolism in a number of 
diseases in which there is limitation of free water 
clearance, water retention, and hyponatremia. In 
patients with cirrhosis, it was found that the anti- 
diuresis resulting from "physiologic" doses of 
pitressin were not more marked and did not last 
longer than the effects produced in normal sub- 
jects. Furthermore, all subjects could excrete free 
water after a water load, albeit in minimal amount. 
The defect in free water clearance in these sub- 
jects was attributable to excessive sodium reab- 
sorption in the proximal tubules, and it was shown 
that they could excrete normal or even increased 
amounts of free water when proximal sodium was 
"delivered" to distal sites with the use of mannitol. 
A similar effect could be produced with infusion 
of sodium chloride and under these circumstances 
the increase of free water occurred even without 
increase of solute excretion. Preliminary results 
have been obtained in patients with cardiac failure 
and a similar mechanism appears to be responsible 
for the defect in free water clearance. 

In Addison's disease there is a defect of free 
water clearance in the presence of large amounts 
of sodium in the urine. It was shown that a 
marked increase in free water clearance could be 
obtained by expansion of total extracellular fluid 
volume with sodium chloride or of intravascular 
volume with albumin. In the absence of any 
steroid therapy, these studies are being pursued 
to determine whether steroid therapy has an addi- 
tional effect and whether hemodynamic changes 
alone would explain the defect in free water clear- 
ance or is antidiuretic hormone hypersecretion 
also involved. 

Study of an additional patient with hypo- 
natremia and bronchogenic carcinoma has shown 
that urinary sodium is, in part, dependent upon 
sodium intake but related to a much greater ex- 
tent to water intake. The findings support the 
view that the syndrome results from sustained in- 
appropriate secretion of antidiuretic hormone and 
does not result from renal or adrenal disease. 

Studies of Calcium Metabolism 

Studies of the essential metabolic abnormalities 
in primary hyperparathyroidism have been con- 
tinued. With the use of a rigorously controlled 



test of the effect of phosphorus deprivation to- 
gether with a low calcium intake, a number of 
new patients with hyperparathyroidism have 
been discovered. The adequacy of phosphorus 
deprivation has been estimated from the extent of 
the decline in urinary phosphorus. When the 
phosphorus deprivation is adequate, patients with 
hyperparathyroidism have shown in all cases a 
rise of urinary calcium and, in most cases, a fall 
of serum phosphorus. The addition of calcium 
loads distinguishes patients with hyperparathy- 
roidism from those with hypercalciuria of other 
origin in that the latter, but not the former, show 
a corresponding increase in urine calcium. The 
addition of glycogenic steroids serves to differen- 
tiate patients with hyperparathyroidism and 
"idiopathic" hypercalciuria on the one hand from 
those with sarcoidosis on the other. In patients 
with sarcoidosis studied thus far, the urine cal- 
cium has fallen with glycogenic steroid. 

Calcium metabolism in known sarcoidosis cases 
has been studied with the use of balance tech- 
niques. These patients have further been sub- 
jected to all the tests currently in use in this labo- 
ratory for hyperparathyroidism. This includes 
the determination of the Tm of phosphorus and 
the response of serum and urine phosphorus to a 
standard calcium infusion during a period when a 
constant diet is given. The balance studies have 
been so designed that the effects of vitamin D (to 
which these patients are said to be hypersensitive) 
and the effects of glycogenic steroid (which are 
said to block the effects of vitamin D in this syn- 
drome) could be assessed separately and together. 
In this way, we are exploring the hypothesis that 
the defect in calcium metabolism in sarcoidosis is 
essentially one of hyperabsorption of calcium, that 
this results from abnormal sensitivity to vitamin 
D, and that the effects are reversible with steroids. 

An attempt to prepare radioactively labeled 
vitamin D is in progress. This is to be used to de- 
termine the fate and distribution of vitamin D. 
The effects of vitamin D will be assessed in a prep- 
aration of rabbit intestine and also in dogs and 
rats that are parathyroidectomized and main- 
tained on calcium alone. 

The defect in the renal concentrating mecha- 
nism in all types of hypercalciuria has been 
studied with renal function techniques. We have 
confirmed that this defect is pitressin resistant. 



82 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



It has been found that it may occur in the pres- 
ence of hypercalcemia or hypercalciuria without 
hypercalcemia. It has been shown to be inde- 
pendent of solute load. A quantitative measure 
of the extent of the defect may be obtained by 
measuring TcH 2 in the manner of Zak, Brun 
and Smith. With this technique it has been 
shown that the defect may be produced in patients 
with "essential" hypercalciuria within 2 weeks by 
high calcium feeding, and that it may be elimi- 
nated by a similar period of calcium deprivation. 
In patients with hyperparathyroidism it was pos- 
sible to return urinary calcium to normal with 
versene and sodium phosphate. With this treat- 
ment the concentrating defect did not improve. 
These studies will be extended to determine 
whether hypercalcemia alone is sufficient to pro- 
duce the defect and to determine the points of 
similarity of this defect with that resulting from 
potassium depletion. 

Studies of Aortic Permeability to Large 
Molecules 

Studies of the movement of protein and lipid 
through arterial walls have been continued in 
rabbits and dogs. Labeled albumin and choles- 
terol have been administered and the rate of accu- 
mulation and loss from the aortic wall has been 
measured as a function of depth of penetration 
and of longitudinal sites in the aorta. Compa- 
rable studies on animals with the hypercholes- 
teremia of hypothyroidism have been instituted. 
The localization of atherosclerosis, as judged from 
these studies on cholesterol deposition, appears to 
depend more upon a decreased rate of cholesterol 
removal from more distal areas than upon in- 
creased rate of deposition. The deposition rate of 
cholesterol and protein in the aorta may best be 
explained, from available data, as a function of 
the circumferential tension in the aortic wall. 

Surgery Branch 

There has been a continuing interest in methods 
for the precise detection and localization of circu- 
latory shunts. The usefulness for this purpose of 
various inert gases, as well as the application of 
indicator-dilution curves, has been previously 
demonstrated. A study of the use of indicator- 
dilution techniques in determining drainage 
pathwaj^s of the pulmonary veins in the presence 



of atrial septal defect, has shown that, by ap- 
propriate injection and sampling, the presence or 
absence of anomalous pulmonary venous drain- 
age can be precisely determined. The validity of 
the method was proved in 29 patients. The injec- 
tion of a radioactive or colored indicator into a 
heart chamber, with sampling at a proximal site, 
makes possible detection of pulmonary or tricus- 
pid valvular regurgitation and estimation of the 
magnitude of the regurgitant flow. Localization 
of the site of origin of a left-to-right shunt by 
means of indicator-dilution curves has, in the 
past, required injections into the left side of the 
heart. The difficulties of this technique were 
found to be obviated by a new method in which 
sampling was carried out within one of the right 
heart chambers following the intravenous injec- 
tion of dye. This simplified technique indicated 
the correct site of entrance of the shunt in all pa- 
tients in whom it was applied. A study was also 
made of the effect of injections of vasopressor 
agents on indicator-dilution curves in patients 
with shunts as well as valvular . regurgitation. 
Whenever a left-to-right shunt was present, its 
magnitude, as measured by indicator-dilution 
curves, was increased when vasopressor drugs 
were given. 

An important advance in the field of indicator- 
dilution methodology has been the adoption fol- 
lowing the work of Clark of ascorbic acid as an 
oxidation-reduction indicator. By means of a 
platinum electrode incorporated within a needle 
the concentration of this reducing substance can 
be recorded continuously without sampling ar- 
terial blood. The principal advantages have been 
in the study of children and experience to date 
demonstrates that the contour of oxidation-reduc- 
tion dilution curves is virtually identical to those 
obtained with blood sampling through a photo- 
electric densitometer. Similarly, the usefulness 
of solutions of cold saline for recording indicator- 
dilution curves has been investigated further. 
Difficulty with the method has been the establish- 
ment of a stable base line, but a new electronic 
circuit has, in pilot studies, apparently obviated 
this. Curves recorded after the injection of cold 
solutions with thermistor sensing in a peripheral 
artery have the same advantages as those de- 
scribed for oxidation-reduction curves. 



NATIONAL HEART INSTITUTE 



83 



It has been shown in several laboratories that 
when a gamma-emitting substance, such as radio- 
active diodrast, is injected intravenously the pas- 
sage of the isotope through the heart can be de- 
tected with a scintillation counter placed over the 
patient's chest. Such curves, recorded in more 
than 100 patients admitted to the service, demon- 
strated that the contour of the precordial dilution 
curve indicates presence or absence of a left-to- 
right circulatory shunt. The method is valuable 
in screening patients and may obviate postopera- 
tive catheterization in patients subjected to opera- 
tion for the correction of such shunts. 

Finally, the principle of isotope dilution has 
been applied in the study of abnormal communica- 
tions between the systemic and portal venous sys- 
tems. In both patients and animals a solution of 
radioactive krypton 8S was injected into the spleen. 
When no abnormal communications existed, the 
appearance of the gas in expired air was greatly 
prolonged. When esophageal varices or a patent 
portacaval anastomosis was present, however, the 
gas appeared immediately and in high concentra- 
tion. It is probable that this method is safer and 
more sensitive than portal venography for asses- 
sing patients with portal hypertension and varices 
both before and after operative treatment. 

An important group of studies has centered 
around the clinical use of the artificial heart and 
lung machine. The instrument itself has been fur- 
ther refined to permit constant observation of the 
oxygen tension in blood returned to the patient, 
precise control of the volume of blood returned to 
the patient, and yet another electronic device has 
been developed which maintains constant the vol- 
ume of blood contained within the patient and the 
extracorporeal circuit. All of these improvements 
have resulted in clinical provisions more closely 
approximating the normal physiologic state. A 
detailed investigation has also been made of the 
bacteriology of the heart-lung machine. These 
studies indicated that in virtually all instances 
bacterial contamination of the apparatus occurs, 
but that it could be minimized by special assembly 
techniques and the installation of bacterial filters 
at all points where room air has access to the 
circuit. 

In the early experience with open heart opera- 
tions, flaccid paralysis of the heart was often 
induced by injection of solutions of potassium 

556044. — 60 7 



citrate into the coronary bed. It was noted in sev- 
eral patients that effective ventricular contraction 
did not resume after cardiac arrest and two 
studies of this phenomenon were undertaken. In 
an experimental study it was shown that left ven- 
tricular function was severely impaired after ad- 
ministration of either potassium citrate or acetyl- 
choline, but that intermittent occlusion of the as- 
cending aorta without a chemical agent had no 
demonstrable effect on ventricular function. In 
15 of 19 patients subjected to potassium citrate ar- 
rest a distinctive type of myocardial necrosis, most 
prominent in the left ventricle, was found. In 
the hearts of 19 other patients in whom this agent 
had not been employed no lesions of this type were 
discernible. These physiological and anatomic ob- 
servations have led to the abandonment of the 
technique of elective asystole in the course of 
cardiovascular operations. Intermittent aortic 
occlusion can be employed in virtually all pro- 
cedures in which aortotomy is not necessary. 
When the aortic valve must be exposed for long 
periods it has been found that effective myocardial 
contraction can be maintained for nearly 2 hours 
by direct perfusion of the left coronary artery. 
This technique is thus employed whenever aortic- 
valve operations are necessary. 

When the heart is divorced from the peripheral 
circulation in the course of cardiopulmonary by- 
pass, there is a unique opportunity for studying 
the effects of various drugs and procedures on 
myocardial contractility, and the central and pe- 
ripheral effects of these agents can be separately 
assessed. In nearly 80 patients a myocardial 
strain gauge arch has been placed at the begin- 
ning of the thoracotomy and in the course of the 
operations injections of various pharmacologic 
agents have been made and their effects on myocar- 
dial contractility studied. It has been found 
that acute digitalization increases the contractility 
of the nonfailing heart. The effects of various 
vasopressor agents have been compared. Norepi- 
nephrine and epinephrine produced identical in- 
creases in cardiac contractile force but vasoxyl 
gave no such response. It has also been possible, 
with the strain gauge arch, to study the effects of 
various anesthetic agents such as fluothance, 
demerol and muscle relaxants, drugs given com- 
monly in the course of cardiac operations. Stud- 
ies of contractile force have further indicated 



84 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the safety of aortic occlusion and coronary per- 
fusion in the course of open procedures. 

The development of methods for catheterization 
of the left side of the heart by the transbronchial, 
the percutaneous and the retrograde arterial 
routes has been described in previous reports. In 
the past year the transseptal method of left heart 
catheterization developed in this laboratory has 
been employed in more than 100 patients. In this 
technique the left atrium is entered by a needle 
passed from the right atrium in the course of right 
heart catheterization. The method provides op- 
portunity for the prolonged measurement of pres- 
sures in the left side of the heart with the patient 
in a comfortable basal condition. No complica- 
tions have been encountered in this limited ex- 
perience. Preliminary experiences also indicate 
that selective angiocardiography, with injection 
into the left atrium through the transseptal needle, 
is a convenient and useful method in the study of 
patients with both congenital and acquired lesions. 

Mitral commissurotomy, in the past, has usually 
been performed with dilatation of the valve with 
the finger inserted from the left atrium or with a 
knife passed from this approach. More recently, 
however, the valve has been opened by means of 
a dilator inserted from the apex of the left ven- 
tricle, the commissurotomy being controlled by a 
palpating finger passed from the left atrial ap- 
pendage. A detailed study of the results of opera- 
tion in patients operated upon by the latter method 
indicates that a superior hemodynamic result al- 
most invariably can be obtained. Twelve patients 
have been operated upon for the second or third 
time for mitral stenosis. In no patient could 
restenosis of the valve be documented and it is felt 
that in most instances the obstruction was residual 
rather than recurrent. In 8 of the 12 patients a 
good hemodynamic and clinical result was ob- 
tained by an effective repeat operation. In the 
course of both open and closed operations for the 
correction of mitral stenosis and mitral regurgita- 
tion, valves have been encountered which are so 
badly damaged that a corrective procedure has 
been either unsatisfactory or impossible. A pros- 
thetic mitral valve, suitable for entire replacement 
oi the diseased valve, has been designed and pre- 
liminary studies of its usefulness have been carried 
out in animals. The present, and most promising 
model is constructed of urethane foam, reinforced 



with plastic mesh cloth. Studies of the effects of 
various plastics and plastic surfaces on the coagu- 
lation mechanisms of the blood have also been 
initiated. Such information will probably be of 
paramount importance in selecting the material 
for fabrication of prosthetic mitral, as well as 
aortic, valves. 

In normal patients or animals, general body hy- 
pothermia at temperatures of 28-30° permits total 
arrest of the circulation for periods of only 10 or 
12 minutes. If the metabolic demands of the body 
could be further reduced by abolition of the thy- 
roid gland this safe period of circulatory interrup- 
tion might be extended. In an investigation of 
this possibility dogs were rendered myxedematous 
by the injection of 1 131 ; survival could be regular- 
ly obtained after 20 minutes of circulatory inter- 
ruption. Another experimental study concerning 
hypothermia has been an evaluation of the effects 
of quinidine on myocardial function. This drug 
is commonly administered during general hypo- 
thermia to prevent arrhythmia. Preliminary re- 
sults thus far indicate that quinidine itself has a 
depressant action on myocardial function. 

The major problem of total replacement of the 
heart is immunologic. Even if means can be found 
to obviate these difficulties, however, many techni- 
cal problems remain. In acute studies in animals 
the transplanted heart is completely denervated 
and this is considered, in most instances, the cause 
of death. An experimental study of the totally de- 
nervated heart in situ is underway and with re- 
finement of the operative technique chronic sur- 
vivors have been obtained. An attempt to 
determine the optimal method for storing an ex- 
cised heart prior to reimplantation is also in prog- 
ress. The comparative value of perfusion with 
blood and various physiologic solutions is being 
studied and attempts are being made to determine 
whether the beating or arrested heart is most suit- 
able for long-term preservation. 

No adequate operation is available for the treat- 
ment of patients with complete atresia of the pul- 
monary artery or true truncus arteriosus. An ex- 
perimental study has been made of methods for 
total replacement of the pulmonary artery by the 
insertion of a plastic graft into the outflow tract 
of the right ventricle and suturing the other end of 
the graft to one or both pulmonary arteries. Death 
has occurred in some animals for technical reasons, 



NATIONAL HEART INSTITUTE 



85 



but in the majority of survivors the prosthesis has 
been proved patent at the time of sacrifice or when 
angiographic studies were carried out. 

Eight patients have now been studied in whom 
obstruction to outflow from the left ventricle was 
caused not by discrete narrowing in the region of 
the valve or subvalvular region, but by massive 
left ventricular hypertrophy of unknown etiol- 
ogy. An attempt has been made to reproduce this 
lesion in the experimental animal. Constrictions 
of the ascending aorta were made either by exci- 
sion of a portion of the wall of the aorta or by 
banding it with a tape of plastic material. Large 
pressure gradients between the left ventricle and 
aorta have been created and serial cardiac cathe- 
terizations are being carried out to determine the 
progression of the lesion. The studies have not 
been in progress long enough to determine if mas- 
sive left ventricular hypertrophy can be induced 
by this technique. 

Considerable effort (in the Section of Cardi- 
ology) has been directed toward elucidating the 
manner in which various hemodynamic factors 
modify the heart's performance. The relation- 
ship between left ventricular end-diastolic pres- 
sure and circumference has been systematically 
investigated in the dog. It was observed that 
with tachycardia, ventricular end-diastolic pres- 
sure rose at a constant end-diastolic circumfer- 
ence. At a constant heart rate, acutely induced 
hypothermia had a similar effect, and it is sug- 
gested that abbreviation of the phase of ventricu- 
lar filling by both of these interventions is re- 
sponsible. On the other hand, deterioration of 
the heart's performance, i.e. acute heart failure, 
resulted in an augmentation of end-diastolic cir- 
cumference at any end-diastolic pressure. This 
appears to be a true alteration in ventricular 
distensibility. However, alterations in aortic 
pressure and cardiac output did not modify ven- 
tricular distensibility. The latter observations 
indicate that myocardial oxygen consumption is 
not primarily dependent on the end-diastolic size 
of the heart. 

In studies of the circulatory responses to acute- 
ly induced hypervolemia in man, it was observed 
that a striking augmentation of cardiac output 
occurred only when the activity of the autonomic 
nervous system had been reduced by ganglionic 
blockade. This investigation shows that in study- 



ing the validity of Starling's law of the heart in 
man, circulatory changes occurring secondary to 
activity of the autonomic nervous system, rather 
than of the heart itself, must be excluded. In- 
deed, in several subjects a consistent relationship 
between ventricular end-diastolic pressure and 
stroke work was observed in the presence of gan- 
glionic blockage and, under these circumstances, 
Starling's law quite clearly operated. In a simi- 
lar investigation of the relationship between left 
ventricular end-diastolic fiber length, end-dias- 
tolic pressure and the force of ventricular con- 
traction in patients with mitral valve disease and 
atrial fibrillation, studied at the time of opera- 
tion, further evidence of the applicability of 
Starling's law of the heart to man was obtained. 

As part of a continuing investigation of the 
pharmacology of digitalis glycosides, it was dem- 
onstrated both in dogs and in patients on cardio- 
pulmonary bypass that these agents are potent 
vasoconstrictors. In addition, a venoconstrictor 
action in the dog was found. These observations, 
when taken together with the studies on myocar- 
dial contractile force in man, described above, per- 
mit a more rational explanation of the effects of 
digitalis on the nonfailing human heart. In a 
study of the effects of acute digitalization on left 
ventricular dynamics, it was shown that the ele- 
vated left ventricular end-diastolic pressure of 
myocarditis may be lowered, but that this did not 
occur in patients with aortic stenosis. It is sug- 
gested in the latter group of patients that the 
hypertrophied ventricular wall alters ventricular 
distensibility and in this manner elevates end- 
diastolic pressure ; the latter then does not reflect 
the presence of myocardial insufficiency, as it does 
in other diseases. Finally, increased digitalis re- 
quirements were demonstrated when hypothyroid 
patients were rendered euthyroid or when euthy- 
roid patients were rendered hyperthyroid. Since 
catechol amine depletion by reserpine blocked this 
antagonism between thyroid and digitalis, the in- 
creased digitalis requirements associated with aug- 
mented thyroid activity are believed to be related 
to increased sensitivity to endogenous catechol 
amines. 

A preparation has been developed in the dog on 
complete cardiopulmonary bypass which permits 
the simultaneous determination of the capacity of 
the vascular bed and of the distensibility of the 



86 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



venous bed. This preparation will permit a study 
on the effects of a variety of cardiovascular re- 
flexes and drugs on these two important hemo- 
dynamic parameters. 

In a continuing study of the factors which 
modify the distribution of blood it has been dem- 
onstrated in normal control subjects that exercise 
results in an increase in intrathoracic blood vol- 
ume and that morphine apparently decreases in- 
trathoracic blood volume. 



Gerontology Branch 

The program of the Gerontology Branch is con- 
cerned with 1) a description of physiological and 
psychological changes that take place with in- 
creasing age in humans and 2) investigations of 
the basic biology of aging. 

HUMAN PHYSIOLOGY 

Longitudinal Studies 

Age differences in physiological and psychologi- 
cal characteristics of normal people still living 
successfully in the community are being evaluated 
by Dr. Shock, Dr. Falzone, and Mr. Norris. Sub- 
jects ranging from 32 to 99 years have agreed to 
return to the laboratory for retesting every 18 
months for the remainder of their lives, so that 
age changes can be observed in individual subjects. 
Retest schedules began in October 1959, so that no 
serial analysis of individual records is possible. 
However, a preliminary comparison has been made 
of some tests on the first 100 subjects with previous 
results obtained on hospital subjects. The outside 
subjects are larger in size than the hospital group. 
However, they do not differ significantly in body 
composition or in basal metabolism from the hos- 
pital subjects. Their pulmonary function is better 
maintained than in hospital subjects and they fail 
to show an age decrement in concentrating ca- 
pacity of the kidney as measured by 12 hours of 
water deprivation, although their 12-hour endog- 
enous creatinine clearance falls with age at a rate 
similar to that of hospitalized subjects. 

In addition to retesting subjects in the series, 
new subjects will be added to the program, with 
special emphasis on men over age 70. A number 
of new tests of intellectual functions and person- 
ality characteristics will be administered to all 



subjects. A special test of attitudes toward aging, 
developed in this laboratory, is being adminis- 
tered to these subjects. 

Renal Studies 

A method for estimating the glomerular clear- 
ance of unbound hemoglobin in the human has 
been developed by Dr. Lowenstein and Dr. Faul- 
stick and hemoglobin/inulin clearance ratios of 
0.028 to 0.065 have been found in preliminary ex- 
periments. The disappearance of the hemoglobin- 
haptoglobin complex from the blood follows first 
order reaction kinetics over the time period of 0- 
120 minutes. Age differences in this function are 
being assessed and the validity of this test as an 
index of the functional capacity of the reticulo- 
endothelial system in the intact human is being 
assessed. 

Comparison between the 12-hour endogenous 
creatinine clearance and short term inulin clear- 
ances have been made in the same subject by Dr. 
Oursler and Mr. Yiengst. Creatinine clearances 
are on the average 30 percent higher than inulin 
clearances, but the correlation is high, so that en- 
dogenous creatinine clearances can be used as an 
index of renal function where intravenous in- 
fusions and bladder catheterizations are impracti- 
cal. Studies of age changes in renal function by 
serial measurements on the same individual will 
be continued. The studies of permeability of the 
glomerular membrane will be continued using in- 
fusions of dextran. 

Body Composition 

The helium chamber method for determining 
body volume has been perfected by Mr. Norris 
and Dr. T. Lundy. Estimates of body composi- 
tion, based on density measurements as well as 
total water content, show a decrement with in- 
creasing age in community residing subjects that 
is of the same order of magnitude as hospital 
patients. Lean body mass, or body water deter- 
minations are being used rather than calculated 
surface area as a basis for the normalization of 
measurements such as basal metabolism. 

Response to Standardized Exercise 

Measurements of age differences in the cardio- 
vascular, respiratory and metabolic responses to 
exercise have been continued by Mr. Norris and 



NATIONAL HEART INSTITUTE 



87 



Dr. Falzone. Maximum work output and rate 
of recovery of vascular and pulmonary displace- 
ments are lower in old than young subjects. Me- 
chanical efficiency is lower in old subjects at high 
and low rates of work, but is essentially the same 
for old and young at intermediate rates. The 
factors involved in the reduced efficiency of the 
aged are being investigated. 

PSYCHOLOGICAL STUDIES 

Although an increase in reaction time with age 
has been previously demonstrated, Dr. M. David- 
off and Dr. G. Suci have shown a linear relation- 
ship between the rate at which information can 
be handled and the age of the subject. This re- 
lationship appears when stimuli are considered in 
terms of information theory. Further investiga- 
tions of this phenomenon in different sense modal- 
ities will be followed. Other studies by Dr. F. 
Hugin and Mr. Norris show that slowing of re- 
sponses with age appears in tasks that involve the 
central nervous system, so that the slowing is a 
reflection of central rather than peripheral 
changes in the nervous system. Dr. W. Surwillo 
is continuing his studies on the relationship be- 
tween EEG frequency and spinal reflex time, to 
determine central effects on reflex times. 

Previous studies, showing that short-term mem- 
ory for visual comparisons is more easily inter- 
fered with in young than old subjects, have been 
extended to include auditory discriminations of 
time intervals by Dr. Davidoff. Other studies of 
memory for verbal material show that the poorer 
memory of older subjects can be improved by re- 
ducing the length of the verbal sequence to be re- 
called and increasing the redundancy or degree of 
relationships between the words in the series. 
Thus, rote memory is impaired to a greater degree 
than memory for logical sequences in older people. 

BASIC BIOLOGY 

Cellular and Comparative Physiology 

The activities of the Cellular and Comparative 
Physiology Section involve (1) the description of 
cellular and organismic changes in humans and 
appropriate experimental animals during the 
aging process and (2) the measurement of the ef- 
fects of environmental and physical manipulation 



on the performance and mortality of experimental 
animals. 

Dr. Bodenstein has demonstrated clearly in his 
studies of regeneration in cockroaches that the ca- 
pacity to regenerate lost parts (e.g. legs) is not lost 
even when the animal would normally no longer 
demonstrate such capacity. Adult cockroaches 
will regenerate legs to replace those amputated if 
supplied with growth hormone through transplan- 
tation of the prothoracic gland from a young 
(molting) animal. This demonstrates that the 
cells of tissues of adult cockroaches still possess the 
capacity to replace lost parts in the proper hu- 
moral environment. 

Dr. Konigsberg has shown that contractile- 
striated skeletal muscles will differentiate from 
embryonic cells in tissue culture and that the proc- 
ess includes the following phases: (a) Cell di- 
vision and multiplication in culture. (The cells 
which will differentiate into muscle are, at this 
stage, not distinguishable from other dividing 
cells in the culture. ) (b ) Cell fusion. During this 
phase, the cells form large multiple nucleated 
fibers or straps. Evidence from time lapse photog- 
raphy and electron microscopy makes it highly 
probable that these are indeed cells with cytoplas- 
mic continuity. During this period, measure- 
ments of DNA per nucleus made in collaboration 
with Dr. Strehler indicate that there is no nuclear 
division following fusion, (c) Differentiation of 
contractile fibers from the multinucleate straps. 
During the early stages of this process striations 
are visible only in glycerinated preparations. 
Later they become visible even in nonglyceri- 
nated preparations observed under phase contrast 
microscopy. 

These findings furnish an ideal tool for the 
study of differentiation, factors affecting it and 
the possible dedifferentiation and redifferentiation 
which have implications regarding the continued 
capacity of cells to furnish replacement parts in 
adults or aged vertebrate animals, provided that 
the factors limiting this process are known and 
modifiable in vivo (as they are, for example, in the 
roach studies). 

Drs. Mildvan and Strehler have continued their 
study of the chemical and physical properties of 
heart age-pigment granules. Approximately 75 
percent of the weight of the particles consists of 
an insoluble material with chemical and infrared 



88 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



absorption characteristics consistent with protein. 
Further elucidation of its nature and of the ad- 
herent as incorporated pigment is being under- 
taken by Drs. Hendley and Strehler. 

Dr. Strehler has continued his study of the effect 
of environmental factors on the longevity of 
Drosophila melanog aster. It appears clear that 
the aging of Drosophila is not a result of denatura- 
tion — since aging does not possess a high activa- 
tion energy. This conclusion follows from the 
fact that flies that have survived high temperature 
shocks sufficient to kill half of them, are not aged 
as measured by their subsequent mortality be- 
havior. Similarly, it has been shown that aging 
in Drosophila is not the result of mutation since 
exposure of animals to 4500 R actually doubled 
their life expectancy. Heavy water, on the other 
hand, in 20 percent or 40 percent concentration, 
reduced the longevity by about a factor of two. 

Studies with Gampanularia flexuosa hydranths 
have demonstrated no decrease with age in the 
following physiological functions : food catching 
ability, rate of ingestion of food, rate of digestion, 
rate of egestion, and amount of food that can be 
handled. Low temperatures extend the life span 
in a fashion similar to that observed with Droso- 
phila melanog aster. X- radiation even in enormous 
doses (50,000 R) does not inhibit the continued 
development of hydranths which has already be- 
gun. Moreover, the animals receiving moderate 
doses of radiation lived twice as long as their con- 
trols — in agreement with the results on Dorsophila 
outlined above. 

Nutritional Biochemistry and Tissue Enzymes 

The determination of various enzymatic activ- 
ities as well as DNA and protein nitrogen in the 
liver, kidney and heart of ten 1-, 3.5-, 12- and 24- 
month-old rats failed to establish a simple clas- 
sification of enzymes into groups which follow 
similar age changes. Dr. Barrows has found that 
although the concentrations of the various en- 
zymes in the tissues of 1-month-old rats were dif- 
ferent from those of older animals, the only 
change which could be associated with senescence 
was the higher cathepsin activity in the liver and 
kidney of the aged rat. No evidence for an im- 
paired protein synthesis in senescent rats was 
found by the depletion-repletion method and only 



slight differences were observed over the age span 
of 3.5 to 24 months. 

A preliminary age study carried out on young 
and old rats subjected to unilateral nephrectomy 
showed similar hypertrophy of the remaining kid- 
ney in both young and old rats. Whereas the in- 
crease in total DNA, d-amino-acid oxidase, and 
alkaline phosphatase approximated the increase 
in organ weight, the total succinoxidase and pyro- 
phosphatase showed a greater increment. The de- 
gree of hypertrophy estimated by any of these 
measurements failed to indicate any age differ- 
ences. 

Future experiments will include further studies 
on oxidative phosphorylation in order to find a 
system which will be adequate for an age study. 

Intermediary Metabolism 

One of the major research activities has been 
concerned with the mechanism of oxidative phos- 
phorylation associated with a-ketoglutarate oxi- 
dation. The oxidative reactions in the electron 
transport chain immediately concerned with the 
synthesis of primary high energy compounds have 
not been identified. Preliminary work led to the 
hypothesis that the critical step might be reduc- 
tion of a disulfide compound to a product with 
vicinal dithiols and simultaneous phosphorylation 
of one of the thiols utilizing the strain energy of 
the cyclic disulfide. Transfer of phosphate to an 
acceptor would lead to a dithiol compound. 
Studies to test the hypothesis are in progress but 
have not yet provided definitive conclusions. 

In a-ketoglutarate oxidation, the primary high 
energy compound is an unidentified acyl enzyme 
complex. The present aim is to identify the com- 
plex and to study the mechanism of its formation. 
Resolution of the a-ketoglutaric dehydrogenase 
complex and purification of the components was 
necessary in order to permit use of stoichiometric 
amounts of highly purified enzyme for direct isola- 
tion of the acyl enzyme complex. One of the re- 
solved components has been purified and shown to 
be a flavoprotein with flavin adenine dinucleotide 
as the prosthetic group. This flavoprotein which 
catalyzes the terminal transfer of electrons from 
the reduced thioctyl to DPN seems to be identical 
with Straub diaphorase. 

There are claims in the literature that the 
morphology and oxidative properties of mito- 



NATIONAL HEART INSTITUTE 



89 



chondria change with age. It is of considerable 
interest in this connection to determine whether 
mitochondria have a defined life span at the end 
of which the entire unit disintegrates or whether 
components within the mitochondria turn over at 
different rates. It is proposed to label three dif- 
ferent components of mitochondria — lipids, pro- 
teins, and cytochrome C — and follow the decrease 
in the labeled component with time. The results 
may be useful in deciding between the two alter- 
native possibilities. 

Biophysics 

Evidence has been found suggesting that the 
amino group on the adenine ring of ATP is in- 
volved in the interaction of ATP with the muscle 
enzyme, myosin, and that this interaction is ac- 
companied by a conformation change of this 
enzyme. Studies in this laboratory have shown 
that Cu ++ , Cal ++ , and Zn ++ interact with myosin 
in a manner superficially similar to that of PCMB 
(parachloromercuribenzoic acid, a sulfhydryl re- 
agent). Furthermore, these metals and PCMB 
are found to interact with myosin during the 
course of incubation with myosin. ATP appears 
to prevent some of this time dependent interaction. 

Cultures of a bleached variety of Euglena gra- 
cilis B have been established and it is hoped that 
these organisms will prove useful as a tool for bio- 
physical and biochemical studies on the effect of 
"age" on single cell systems. Preliminary results 
have shown that such cultures may be kept well 
over one month at constant cell density. Experi- 
ments have now begun to study the effects of 
aging under various well-defined conditions on 
subsequent growth. Since these organisms are 
sensitive to steroid hormones, Dr. Buetow has 
tested their response to vitamin D, and has found 
that growth appears accelerated by this vitamin. 
This is the first time an effect of this vitamin has 
been found in other than a mammalian system. 

After the biochemical characterization of the 
mitochondria from Euglena has been completed, 
studies will be undertaken to determine the effects 



of aging on a variety of mitochondrial functions, 
such as stability, turnover, permeability, etc. 

Molecular Biology 

Important findings in the elucidation of the 
structure of catalase are that the four heme 
groups are symmetrically placed in the molecule, 
and that they can be reversibly removed from the 
molecule without affecting their site of attach- 
ment to the protein. 

A method is being developed for detecting the 
position of nucleosides in a nucleic acid chain by 
selective complex formation with metal ions. 

Catalase that has been cleaved into quadrants, 
each containing one heme, has no anomalous ro- 
tatory dispersion, indicating that the heme is sym- 
metrically surrounded by protein ; intact catalase, 
on the other hand, containing four hemes, has an 
asymmetric iron atom. The heme has been re- 
moved from catalase, yielding the apoenzyme ; the 
latter can aparently be recombined with hemin to 
reform the catalase molecule. 

Horse hemoglobin is split along different axes 
by acid and base treatment. Human hemoglobin, 
like horse hemoglobin, has asymmetric iron in the 
reduced, oxidized, and oxygenated forms. 

Vitamin B i2 exhibits anomalous rotatory disper- 
sion due to the presence of an asymmetric cobalt 
atom. The rotatory dispersion curve is unaffected 
by substitution for cyanide on the cobalt atom, and 
it is not greatly affected by reduction of cobalt. 
The rotation is markedly influenced, however, by 
changes in the three-dimensional structure of the 
molecule. 

A correlation has been made between the elec- 
tronic configuration of transition metal ions and 
their ability to catalyze the aconitase and enolase 
reactions. It has been shown that nickel, cobalt, 
and iron catalyze the aconitase reaction in the ab- 
sence of enzyme. The mechanism of formation of 
the Schiff base intermediates in transamination 
reactions has been further elucidated by the find- 
ing that a carbinol amine intermediate is not 
formed in such reactions. 



NATIONAL INSTITUTE OF ARTHRITIS AND 
METABOLIC DISEASES 



BASIC RESEARCH 

INTRODUCTION 

For the NIAMD to fulfill its assigned mission, 
its scientists are impelled to conduct research over 
a wide spectrum, ranging from the purest and 
most basic to the applied and immediately practi- 
cal. Much has been written about the distinctions 
between these types of research and the purported 
merits of the one over the other. It is not our 
purpose at this time to enter into these contro- 
versial matters, other than to clarify the reasons 
why it is that both extremes, as well as all the 
intermediate shades between the practical and the 
pure, are legitimate areas of inquiry for scientists 
at this institute. 

The benefits of applied research and develop- 
ment are likely to be immediate and immediately 
apparent. A new treatment for a disease, a new 
diagnostic procedure, a new prophylaxis, is a justi- 
fication in itself, and the quest therefor requires no 
additional defense. Such research is characterized 
by the fact that the end is in view from the outset, 
and the motivation of the investigator is in major 
part this end in view. The research, if successful, 
has immediate consequences of assistance to the 
patient and to the doctor. A disease is more pre- 
cisely diagnosed, more effectively treated, and 
more uniformly prevented than formerly. If the 
problem attacked is of sufficient magnitude, such 
research may markedly benefit the public as well 
as the private health of the Nation and the world. 

It is, however, the history of science that nearly 
every practical advance is sooner or later rendered 
obsolete by yet another succeeding development. 
The new drugs of yesterday are supplanted by the 
new drugs of tomorrow, the diagnostic tests of 
yesterday are often only of historical interest to- 
day. In this sense, the results of applied research, 
although possibly of enormous contemporary im- 
portance, are largely to be evanescent. It is true of 

556044 — 60 8 



course, that the applied research of today may 
serve as a basis for future developments. This 
however is a by-product and insofar as it occurs, 
the earlier research should be regarded as basic in 
character. 

The outstanding characteristics of basic re- 
search are its purpose and its motivation. Its pur- 
pose is simply to add to man's knowledge and un- 
derstanding. No end other than this is in view. 
The motivation of the scientist engaged in such re- 
search is quite simple. It becomes increasingly 
clear that curiosity about nature is the major driv- 
ing force of our most productive investigators in 
biomedical laboratories. In contrast to the im- 
mediate, often transient, effects of applied re- 
search, the fruits of pure research have a perma- 
nence about them. In a changing world there 
are few things so likely to endure as are the results 
of good basic research. Their importance lasts 
through the ages and may at any time give birth to 
further extension, both pure and applied. 

It is in the nature of science to be both recorded 
and cumulative. All scientists depend heavily 
upon the researches of their predecessors, and 
doubtless this is what Newton had in mind when 
he wrote, "If I saw farther, it was because I stood 
on the shoulders of giants." The additions to 
man's knowledge, accomplished by the scientist of 
today, are the foundations upon which scientists 
of the future, both pure and applied, must build. 
In these terms, practical benefits from pure re- 
search are essentially inevitable, even though not 
necessarily anticipated when the research was un- 
dertaken. 

We review with pride our research accomplish- 
ments for the calendar year 1959. The signifi- 
cance of the more applied aspects of our program 
is self-evident. Fully as significant are the results 
of the more basic aspects of our program. The 
conduct of basic research requires no apologies. 
It is a good thing to do for its own sake and we 
are happy to be in a position to support so many 

91 



92 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



fundamental investigations in natural sciences at 
NIAMD. The enduring fruits of basic research 
are the highest rewards of the scientific career. 

The Blessing (and Curse) of Bigness. NIH 
is probably the largest concentration of science 
and scientific manpower in biomedical research 
in the world today. This fact carries with it cer- 
tain advantages as well as some disadvantages. 
Furthermore, some of the benefits are more imag- 
inary than real. Because of its great size, NIH 
•can economically support vast central sepwices 
such as an animal-production facility, extensive 
shops, and a fine library. It is economically fea- 
sible for NIH to engage in the purpose or con- 
struction of costly equipment which might be 
beyond the reason of a smaller institution. 

It is noteworthy that, in the past, but a very 
small fraction of the major accomplishments of 
NIAMD have been directly dependent upon these 
purported advantages. To a far greater extent 
these accomplishments have depended upon the 
ideas and skills of the individuals who comprize 
the scientific and clinical staffs. The fraction of 
prominent research conducted at Bethesda which 
would have been impossible elsewhere for want of 
physical plant is not great, and this is also true 
of this year's results, reported in what follows. 

Furthermore, there is a necessary price paid for 
the benefits of bigness. This is to be seen in the 
increased difficulties in communication, at all 
levels. The investigator in so large an institution 
loses contact with the administrator responsible 
for budget and policy decisions. He finds it in- 
creasingly difficult to maintain intimate contact 
with the shop that builds his equipment or the 
agency which renders some essential service to his 
project. Most important, with almost 1,000 sci- 
entists on the campus, each individual scientist 
can be familiar with only a small fraction of what 
is going on in other laboratories. 

There is, on the other hand, one blessing of 
bigness, which, for certain investigators, is over- 
riding. Among the many scientists about Be- 
thesda, virtually all research disciplines are 
represented. Very high degrees of skill are to be 
found in physics, mathematics, chemistry, the bio- 
logical sciences, and medicine. In the present era 
of research, the discovery that there are unplowed 
fields between established disciplines, that these 
fields contain pay dirt, has favored multidisci- 



plined approaches to problems of interest. Some, 
but certainly not all of our more effective investi- 
gators, have realized and taken advantage of this | 
situation. They have increased the scope of their 
effectiveness by the establishment of collaborations 
with others of diverse background and experience. 
Such collaborations are likely to be exceptionally 
productive. It often appears that the whole is far 
greater than the sum of its parts. 

It must be borne in mind that not all scientists 
are naturally collaborative. Some of our best 
scientists, like most artists, work most effectively 
by themselves. Others prefer to acquire satellite 
scientists about them. Still others prefer to ex- 
plore interdisciplinary areas in conjunction with 
scientists of other skills. It is to the last group 
that the size of NIH is a particular blessing. 

Recruitment and Adjustments of Program. A 
number of additions to staff have been made. In 
the clinical area, a group interested in metabolic 
diseases of infancy and childhood has been col- 
lected. A part of the interests of this group is in 
cystic fibrosis, a pediatric problem of growing 
importance. A start has been made also in the 
area of diseases of the gastro-intestinal tract. In 
the laboratory area, it has been in the physical- 
mathematical specialties where most recruitment 
has occurred. Personnel in these specialties are 
in low supply and in high demand, making re- 
cruitment particularly difficult. A number of 
people of high ability have, however, been found 
and brought to Bethesda. 

The recent retirement of Dr. Ralph Wykoff, 
and the imminent retirements of Dr. Ralph Lillie 
and Nathan Eddy, have created problems. For 
each of these vacancies, candidates have been 
considered and tentative solutions reached. It is 
hoped that before next year's report, a happy 
resolution of each of these problems will have 
been reached. 

Laboratory of Nutrition and Endocrinology 

One of the primary goals of the laboratory is 
to investigate the biochemical, physiological, and 
histological changes that occur in animals asso- 
ciated with dietary or endocrinological altera- 
tions. Such studies should eventually indicate 
how the nutrients and hormones carry out their 
essential functions. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



93 



NUTRITION 

Factor 3. One of the more important scientific 
contributions made by this laboratory was the rec- 
ognition that selenium is the active component of 
Factor 3. Eats developed a dietary -liver necrosis 
on a diet devoid of vitamin E, cystine, and Factor 
3. In 1957, it was shown that the necrosis could 
be prevented by the addition of 4 to 6 ,/xg. of 
selenium (selenite) to 100 gm. of diet. The natu- 
rally occurring form of Factor 3 is 3 to 5 times 
as active as an equivalent amount of inorganic 
selenium. The chemical properties associated 
with Factor 3 activity have been elucidated from 
studies of synthetic organoselenium compounds. 
One of these compounds, a racemic diselenocar- 
boxylic acid, is half as active as Factor 3 on the 
basis of equivalent selenium contents. 

Earlier work showed that most of the activity 
of cystine in protecting rats against dietary liver 
necrosis was traceable to contamination with a 
small amount of biologically active selenium. 
More recent work shows that the sulfur amino 
acids when added to the necrosis-producing diet 
delay the onset of the liver disease but do not pre- 
vent it. The addition of selenium-free cystine, 
homocystine, or methionine reduced to one-tenth 
the level of vitamin E required for the prevention 
of dietary-liver necrosis. When selenite replaces 
vitamin E, the sulfur amino acids reduce the level 
required for the prevention of liver necrosis by 
30 to 50 percent. 

Studies of the metabolic disturbances associated 
with dietary-liver necrosis showed that liver slices 
of rats with necrotic livers are unable to maintain 
a normal rate of oxidation. However, the mito- 
chondria prepared from such livers show no de- 
cline in respiration when incubated with various 
members of the tricarboxylic-acid cycle. Suc- 
cinate was the exception in that the mitochondria 
showed a respiratory decline when DPN was 
added to the system. Homogenates of the above 
livers show a respiratory decline similar to that 
of the liver slices with a-ketoglutarate or suc- 
cinate as substrate. Dietary Factor 3 (as selenite) 
is without effect on these systems suggesting that 
Factor 3 and vitamin E participate in different 
pathways of intermediary metabolism. 

The mitochondria from the deficient rats show 
more succinate cytochrome-c reductase and oxal- 



acetic-decarboxylase activity than those from 
vitamin E-supplemented animals. A possible ex- 
planation for this anomalous finding is that the 
mitochondria from the deficient animals may per- 
mit greater access of substrate to enzymes due 
to structural change. Suggestive evidence for the 
presence of changes in the mitochondria comes 
from the increased swelling of the deficient mito- 
chondria when suspended in a hypotonic medium. 
The addition of adenosine monophosphate to the 
preceding system overcomes the increased swelling 
of the deficient mitochondria. 

Further study of the "swelling" of mitochon- 
dria confirmed observations of others that oxida- 
tive phosphorylation prevents it. The confusion 
existing in the literature as to the action of 2,4- 
dinitrophenol (DNP) in this system was resolved 
when it was shown that this compound could 
both prevent and accelerate swelling under differ- 
ent conditions. In media where phosphorylation 
is not possible, DNP protects ; in a phosphorylat- 
ing medium where adenosine monophosphate is 
the acceptor, DNP accelerates swelling; when 
adenosine diphosphate is the acceptor, DNP pre- 
vents swelling. A possible explanation for the 
peculiar behavior of DNP is that it acts on 
adenylate kinase at the surface of the mito- 
chondria. 

Vitamin E. Closely allied to the dietary-liver 
necrosis is exudative diathesis which develops in 
chicks fed a diet similar to that used in the rat 
work. In 1957, when the activity of Factor 3 
was shown to be associated with selenium, it was 
observed that trace amounts of this element would 
prevent or cure exudative diathesis. Further- 
more, when selenium was added to a vitamin 
E-free ration on which exudative diathesis was 
routinely produced, the animals now developed 
encephalomalacia. Thus, it became possible to 
explain, on the basis of the amount of selenium 
in the diets, the appearance of different vitamin 
E-deficiency syndromes reported by various in- 
vestigators. 

Additional work showed that chicks could be 
reared to adulthood on diets free of vitamin E 
provided the level of unsaturated fatty acids there- 
in is very low. These vitamin E-free chicks grew 
at a normal rate and, following artificial insemina- 
tion, layed eggs which were shown to be fertile. 



94 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Since no tocopherol could be detected in the tis- 
sues of these birds, it is suggested that vitamin E 
plays no role in the intermediary metabolism of 
chicks. 

Other workers observed alterations in the serum 
proteins of vitamin E-deficient chicks suggesting 
that these changes might contribute to the edema 
seen in exudative diathesis. Studies in this labo- 
ratory show a decrease in albumin : globulin ratios 
in the blood of the deficient chicks but these 
changes were not great enough to account for the 
edema. The most prominent changes occur in the 
chicks showing spontaneous recovery where, after 
the recovery, increases appear in a 2 -, a 3 -, /?- and y- 
globulins. 

As work progressed on the vitamin E-deficiency 
in chicks, it became obvious that some of the older 
reports on the pathological changes attributed to 
this deficiency were complicated by a concomitant 
deficiency of vitamin A. The latter deficiency 
would be hastened by a simultaneous dietary vita- 
min E deficiency. Work over the past few years 
has shown that in vitamin A deficiency, chick 
brains show scattered pyknotic neurons located in 
the optic tactum and Purkinje-cell layer of the 
cerebellum. In vitamin E-deficiency, large necrot- 
ic areas occur in the cerebellum and occasionally 
in other parts of the brain. A combined vitamins 
A and E deficiency produced many subcellular 
areas, especially in the frontal lobe of the brain. 

A few years ago, a report appeared which sug- 
gested that vitamin E played an integral role in 
enzymatic oxidation. This was based on the ob- 
servation that extraction of a cytochrome reduc- 
tase preparation with a fat-solvent reduced the 
enzymatic activity which could be restored by the 
addition of Vitamin E. Work in this laboratory 
showed that the first action of the solvent is to 
combine with proteins thereby disrupting electron 
transport. Vitamin E appears to desorb the sol- 
vent. The vitamin is not alone in showing this 
effect especially with aged preparations since sev- 
eral hydroxylated antioxidants such as Santoquin 
and 5-pentadecyl resorcinol are also highly active. 

Previous work on the metabolism of vitamin E 
has been handicapped by the analytical procedures 
which result in a poor separation of the vitamin 
and interfering substances. A new technique has 
been developed which permits excellent chromato- 
graphic separation of vitamin E by means of a 



column of basic zinc carbonate, aluminum oxide, 
and celite. 

Vitamin B 12 and Folic Acid. Some years ago, it 
was shown in this laboratory that the poor growth 
secured with normal chicks fed a diet containing 
large amounts of fat and minimal amounts of 
methionine, could be overcome by adding vitamin 
B 12 to the diet. With this diet it was observed 
that a vitamin B 12 deficiency had no effect on car- 
cass composition in so far as protein, ash, and 
moisture were concerned. This finding suggests 
that the report of others on the importance of vita- 
min B 12 in protein synthesis does not explain the 
physiological function of the vitamin in so far as 
the chick is concerned. It has been shown that the 
diets used in the above work must be deficient in 
methionine— a similar diet deficient in arginine 
does not show growth inhibition when fat is added. 
Apparently there is a specific vitamin Bi 2 -methio- 
nine relationship in the chick which is sensitive to 
high levels of fat in the diet. 

The vitamin B 12 -deficient chick is similar to the 
rat in that each species excretes large amounts of 
formiminoglutamic acid in the urine. Vitamin 
B 12 supplementation of the deficient chicks for 
several days produced a reduction in the excre- 
tion of this histidine metabolite. Supplementing 
the deficient diet for one day with methionine 
produced an immediate drop in formiminoglu- 
tamic acid excretion which was followed by levels 
of excretion greater than those seen in the pre- 
supplementation period. 

Earlier work in this laboratory showed that rats 
deficient in both folic acid and vitamin B 12 ex- 
crete large amounts of formiminoglutamic acid. 
The urinary excretion of this compound is in- 
creased when histidine is fed to the deficient rats. 
If ethionine (a methionine antagonist) is fed to 
these rats, the excretion of formiminoglutamic 
acid is reduced but the excretion of urocanic acid 
is increased. The ethionine inhibits the synthesis 
of urocanase, a deficiency of which blocks the me- 
tabolism of histidine at a step prior to the block 
induced by the deficiency of folic acid or vita- 
min B 12 . 

Although there is an apparent interrelationship 
between vitamin B i2 and folic acid, it is not a di- 
rect one. Vitamin B 12 is not involved in the acti- 
vation of folic acid since rats deficient in the 
former can still convert folic acid to tetrahydro- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



95 



folic acid and related compounds which are as- 
sumed to be the metabolically active forms. 

Additional evidence on the role which vitamin 
B12 may play in physiological processes comes 
from the purification of a protein which contains 
the vitamin firmly attached to it. This complex 
is required for the synthesis of methionine from 
homocysteine and serine by extracts of E. coli. 

Although it is assumed that folic acid occurs 
as the citrovorum factor, evidence has been ac- 
cumulated in the laboratory which suggests that 
the naturally occurring form of folic in the liver 
has not hitherto been characterized. Extracts con- 
taining unknown forms of pre-f olic acid that can 
be converted to citrovorum factor by liver enzymes 
can be prepared from horse liver without enzy- 
matic treatment. One of these pre- folic acid com- 
pounds has been partially purified. It can be con- 
verted successively to tetrahydrofolic acid and 
then to citrovorum factor by two separate enzyme 
systems. The first of these systems requires 
catalytic amounts of flavin adenine dinucleotide 
and a suitable hydrogen acceptor. The second re- 
quires formylgiutamic acid and a liver enzyme 
preparation which forms citrovorum factor (N-5 
f ormyltetrahydrof olic acid) . 

Germ-Free Animals. Germ-free animals ap- 
pear to be the only means whereby unequivocal 
answers can be secured to such problems as the 
contribution of the flora in the gastrointestinal 
tract to the nutrition of the host. Studies are 
under way on the mechanism whereby antibiotics 
and large amounts of ascorbic acid in the diet re- 
duce the animal's requirement for the B vitamins. 
Unless conventional rats receive pantothenic acid 
in their diets, they do not grow. When panto- 
thenic acid is replaced by either 0.5 percent as- 
corbic acid or 100 mg. percent penicillin, the rats 
grow almost as well as those receiving pantothenic 
acid. There has been some controversy as to the 
exact mechanism whereby ascorbic acid and the 
antibiotics spare the animal's requirement for the 
B vitamins. When the above work was repeated 
with germ-free rats, no sparing effect of vitamin 
C or penicillin was seen, indicating that the action 
of the latter compounds was mediated through the 
intestinal flora. 

A severe vitamin K deficiency develops in germ- 
free rats fed rations free of this vitamin. Con- 



ventional rats can maintain a normal blood- 
clotting time without any dietary source of this 
vitamin. On a low-fat diet, the vitamin K-defi- 
cient, germ-free rats died in 30 days. Increasing 
the fat in the ration decreased the survival time. 
A normal clotting time develops within 24 hours 
after the germ-free rats become contaminated on 
removal from the tank. It has been shown that 
sulf aquinoxaline functions not only as a bacterio- 
static agent but also as a vitamin K antagonist. 
When germ-free rats are fed vitamin K-free diets 
containing sulfaquinoxaline, they develop hemor- 
rhages within 2 weeks instead of the 4 weeks re- 
quired on the basal diet. 

Another method of studying the contribution of 
the intestinal flora to the rat's nutritional require- 
ments is to work with animals having a cup over 
their anuses in which the feces are collected. Such 
rats excrete almost one and a half times as much 
feces as the rat that has access to its own feces. 
These "cupped" rats develop signs of a folic-acid 
deficiency when a diet low in this nutrient is fed. 
Rats that have access to their feces show a normal 
blood picture on such a diet, suggesting that the 
folic acid synthesized by the bacteria in the gas- 
trointestinal tract of the rat is absorbed only after 
the animal consumes its feces. The above explana- 
tion is limited by the observation of a change in 
the intestinal flora as a result of the "cupping." 
The Lactobacilli count in the feces of rats with 
tail cups was only 0.1 percent that seen in their 
feces prior to cupping. 

Protein. Studies have been initiated to deter- 
mine whether the dietary deprivation of protein 
preferentially changes the activities of certain 
components of an enzyme system. Preliminary 
observations suggest that protein deprivation for 
30 days in the rat produces only a slight decrease 
in succinic dehydrogenase, no change in the cyto- 
chrome-b-cytochrome-Cx complex, a 70 percent re- 
duction in cytochrome oxidase, a slight reduction 
in succinate-cytochrome-c reductase, and a 40 per- 
cent reduction in the activity of the overall suc- 
cinic-oxidase system when measured by one meth- 
od and no change when measured by a second 
method. 

Obesity. Weanling rats fed the high-fat, obes- 
ity-producing diet show an increase in carcass fat 
over the lean controls as early as the fifth week 



96 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



even though the body weights of the two groups 
are very similar. The livers of the obese rats are 
about 50 percent heavier than those in the lean 
controls but the percentages of protein, fat, and 
water are not changed. Similar findings hold 
true for kidneys and hearts. The adrenal glands 
in the obese rats are larger and have a higher per- 
centage of neutral fat and vitamin C than the 
lean rats while the concentrations of cholesterol 
and phospholipid are the same, 

The obese rats appear to absorb vitamin Bi 2 
to a greater extent than the controls as shown by 
higher plasma and liver levels. These increases 
occur in the obese rats even though the dietary 
levels of the vitamins are the same. Another 
peculiarity of the obese rat is the marked pro- 
teinuria which occurs in both males and females. 
These proteins are mainly albumin and /?-globu- 
lin. The proteinuria is reduced by the addition 
of an inert filler (Solkafloc) to the high-fat diet. 

Guinea Pigs. Work has been completed on the 
tryptophan requirement of the guinea pig which 
shows that even though maximum rates of growth 
are secured with 0.03 percent L-tryptophan in the 
ration, cataracts develop. Eyes appear to be nor- 
mal only when the dietary tryptophan level is 
raised to 0.1 percent. The above studies will be 
facilitated by a newly developed diet in which the 
protein is replaced by an amino-acid mixture that 
permits as good growth as does the protein diet. 

Rabbits. It has been assumed that rabbits do not 
require any dietary source of thiamine. That this 
may not be strictly so is suggested by work in 
which 3- to 4-week-old rabbits are put on a thia- 
mine-free diet. On this diet, the animals grow 
almost as well as the controls receiving supple- 
mental thiamine. However, about 40 percent of 
the rabbits on the deficient diet show locomotor 
difficulties after 3 or more months of feeding. 
The urinary excretion and fecal liver and brain 
content of thiamine were lower in the animals on 
the deficient diet than in the controls. 

ENDOCRINOLOGY 

Glucose-Tolerance Factor. An interesting ob- 
servation made during the past year was the rec- 
ognition that trivalent chromium is an integral 



part of the glucose-tolerance factor (GTF). A 
single dose of 20 to 50 mg. per 100 gm. rat will 
increase the removal rate for intravenously in- 
jected glucose from 2.8 to 4.8 percent per minute. 
The activity of chromium is confined to the triva- 
lent form. The potency varies from compound to 
compound with the very stable coordination com- 
plexes of chromium being practically inactive. 
When adipose tissue from rats on a GTF-supple- 
mented diet was incubated in a medium contain- 
ing insulin it removed almost twice as much glu- 
cose as did the tissue from the deficient animals. 
The addition of 0.1 fig. of trivalent chromium to 
the flask containing the deficient tissue increased 
the uptake of glucose and its incorporation into 
fat threefold as shown by means of labelled glu- 
cose experiments. Preliminary evidence suggests 
that in stabilized diabetic rats (alloxan), trivalent 
chromium reduced the fasting blood-sugar level 
from 250 to 125 mg. and abolished ketonuria. 

Experimental Diabetes. Diabetes produced in 
the rat by removal of 99.5 percent of its pancreas 
results, within 2 hours of the operation, in elevated 
blood levels of glucose, ketone bodies, and tri- 
glycerides. The increases in the latter two sub- 
stances occurred before diabetic blood-sugar levels 
were reached. The increase in blood lipids was 
accompanied by excessive accumulation of fat in 
the liver and kidneys. 

The level of fat in the liver of the diabetic rat 
is linearly related to the level of blood-ketone 
bodies. Removal of various endocrine glands 
showed that glucocorticoid hormone is the only 
one required for the development of fatty livers 
and ketosis in the diabetic rat. Growth hormone 
has no effect and ACTH is ketogenic only if the 
adrenal glands are intact. The adipokinetic and 
ketogenic actions of glucocorticoids can be over- 
come with insulin. The peripheral utilization of 
D(-)-/?-hydroxybutyrate is reduced 60 percent in 
the pancreatectomized rat when insulin is with- 
held. Acetoacetate utilization is also reduced but 
to a lesser extent. On these bases, it appears that 
ketosis in the insulin-deficient rat results from an 
increased production and a decreased utilization 
of ketone bodies. 

An elevated blood-glucose level appears to be 
the primary factor regulating the secretion of in- 
sulin from the pancreas. Peripheral blood of 






NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



97 



dogs during fasting contains about 37 microunits 
of insulin per ml. of plasma. Administration of 
glucose increases the output of insulin by the 
pancreas five- to ten-fold. Pituitary hormones and 
the antidiabetic drugs such as tolbutamide do not 
stimulate the pancreas to release additional 
insulin. 

Pituitary Hormones. — Purification of the thy- 
roid-stimulating hormone (TSH) has progressed 
to the point where it is evident that activity is as- 
sociated with several different proteins. The frac- 
tion prepared from TSH-producing pituitary 
tumors in mice has been shown to be free of ex- 
ophthalmogenic and luteinizing activity. The 
thyrotropic effects of the mouse as well as human 
TSH preparations can be neutralized by anti-sera 
to bovine TSH which suggests a lack of species 
specificity in TSH. When chicks are treated with 
TSH, their thyroid glands show a reduction in 
sialic acid (a constituent of thyroglobulin) which 
parallels the depletion of iodine. 

Preliminary studies show that in pigeons, 
from which the pars distalis of the hypophysis was 
removed, treated with insulin, the weight of the 
adrenal gland increases markedly over that of the 
hypophysectomized controls. 

Studies are under way to perfect an assay for 
ACTH levels in the blood. This is based on the 
measurement of corticosterone in adrenal-vein 
blood of hypophysectomized rats following the in- 
jection of the hormone. As little as 50 microunits 
of ACTH produces a significant increase in the 
output of corticosterone by the adrenal gland. 

Steroids. Analytical methods have been de- 
veloped which have permitted the measurement 
of A 5 - 17-hydroxy pregnenolone, A 5 -pregnenetriol, 
and dehydroepiandrosterone in urine. Six 
patients with adrenal carcinoma exreted no hy- 
droxyprenenolone but large amounts of both preg- 
nenetriol (3 to 50 mg. per day versus 0.2 mg. in con- 
trols) and dehydroepiandrosterone (6 to 90 mg.) 
Pregnenetriol is thus established as an abnormal 
steroid metabolite characteristic of adrenocortical 
malignancy. 

Midbrain Lesions and Endocrine Activity. 
The effect of the central nervous system on meta- 
bolic and endocrinoloffic reaction was studied in 



dogs with transections of the upper midbrain. 
These dogs show a depression of the hypothalami- 
copituitary activating system whereas in animals 
where the transection was not successful, the op- 
posite effect was noted, namely a marked increase 
in urinary corticoids following a stressful ex- 
perience. Spinal-cord transection in the dog re- 
sults in a marked increase in urinary creatine ex- 
cretion. By means of isotopically labelled com- 
pounds, it has been established that the source of 
the increased urinary creatine is muscle creatine. 



Laboratory of Pharmacology & Toxicology 

Shock and Infection. The problem of delayed 
deaths following extensive burns is receiving 
major attention. In the Peru Project, where eval- 
uation of oral saline solution and plasma therapy 
for burn shock is being carried out, over half of 
the late deaths are due to Pseudomonas and 
Staphylococcus septicemias. Antibiotic therapy 
is not effective, and studies in burned mice indi- 
cate that a lowering of host resistance is important 
in the genesis of these infections. In mice, gamma 
globulin has been highly effective prophylactically 
and for the past two years it has been used on 
alternate cases in the Peru Project. The results 
have been encouraging, in that a significant de- 
crease in septicemias occurred in the treated group. 

Since Pseudomonas is the principal infection, 
a highly potent rabbit immune serum has been de- 
veloped. This is 800 times as potent as gamma 
globulin in mice, and is effective therapeutically as 
well as prophylactically. This is being tried in 
established Pseudomonas septicemias in the Peru 
Project. 

In the Peru Project, serum albumin is also being 
compared with plasma in the treatment of shock. 
Apart from comparing its efficiency in shock, it 
will also reveal whether the absence of antibodies 
will predispose to these septicemias. 

In the field of experimental leprosy, conditions 
for the growth of Mycobacterium muris in tissue 
culture of monocytes have been established. Cul- 
tures can be maintained 1 to 2 months, and afford 
a means of in vitro study of the organism, and of 
assay of chemotherapeutic agents. 

Calcium in Nerve and Muscle Function. Fur- 
ther evidence has been obtained that the calcium 



98 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



ion may be an essential factor in muscle contrac- 
tion. Caffeine contraction of striated muscle, in 
concentrations low enough not to cause membrane 
depolarization, increases Ca 45 influx and outflux 
threefold. Increases are also seen after potassium 
depolarization, but it has been shown that caffeine 
affects calcium sites in the membrane distinct from 
those affected by membrane depolarization. 

The behavior of veratrum alkaloids on a mono- 
molecular surface film of stearic acid has been 
studied in an attempt to correlate physico-chem- 
ical behavior with pharmacological action. The 
pharmacologically active alkaloids penetrate and 
interact with the film, and orient both horizontally 
and vertically at the interface. The inactive alka- 
loids show only weak penetration and orient only 
horizontally. 

Amine Metabolism. The biosynthesis of sper- 
midine by purified enzymes from E. coli has been 
accomplished, according to the scheme: 

I. Methionine + ATP-»adenosylmethionine 
+ PP + P 

II. Decarboxylation of adenosylmethionine 

III. Putrescine + Il^spermidine + thiometh- 
yladenosine 

Enzyme I has been purified 2,000-fold and re- 
quires only magnesium. Enzyme II has been 
purified 20-fold, and requires magnesium. En- 
zyme III has been purified 1,000-fold, with no 
cof actor demonstrable. All of the products have 
been characterized. Spermine and spermidine are 
widely distributed in plant and animal cells and 
it is believed they have an important function. 
This elucidates their formation, as well as that of 
thiomethyladenosine . 

Decarboxylated adenosylmethionine has been 
prepared by chemical synthesis, and the synthetic 
product shown to be active as a substrate for III. 

Glutathione-Polyamine Conjugate. A high 
percentage of the glutathione in E. coli cells was 
found to be present as a conjugate with spermi- 
dine. It has been characterized by its behavior on 
ion-exchange resins and paper chromatography, 
and by identification of the amino acids and 
spermidine after hydrolysis of the isolated com- 
pound. When spermine, which is not normally 
present in E. coli, is added to the medium, a simi- 
lar conjugate is formed with spermine. 



Acetylation of Polyamines. Taking advantage 
of the differential absorption spectra of dinitro- 
fluorobenzene derivatives of primary and second- 
ary amines, a method has been developed which 
is useful for the assay of various amines, includ- 
ing the acetyl derivatives of the various poly- 
amines. Monoacetyl putrescine and two isomeric 
forms of monoacetyl spermidine have been iso- 
lated from E. coli cells. They have been further 
characterized by behavior on ion-exchange resins 
and paper chromatography, and by acetate and 
amine assays on hydrolyzed samples. 

Metabolism of Histidine and Related Com- 
pounds. Histidine is an essential amino acid and 
enters into many important metabolic relation- 
ships. Carbon-2 of the imidazole ring enters the 
"one carbon" pool which involves folic acid and 
vitamine B 12 in its metabolism. Five steps have 
been shown in this degradation : 

I. Histidine— »Urocanic acid+NH 3 

II. Urocanic— >i ormimino - glutamic acid 
(Figlu) 

III. Figlu -ftetrahydrofolic (THF)^Form- 
iminoTHF + glutamic acid 

IV. FormiminoTHF^5-10 methenylTHF + 
NH S 

V. 5-10 methenylTHF^lO formylTHF 
Enzymes have been purified from bacteria or 

animal tissues which catalyze each of these steps. 
Enzyme II has been purified 100-fold and III and 
IV, 1,000-fold. The kinetics and requirements of 
the latter have been characterized. The reversi- 
bility of V at neutral pH is important since 5-10- 
methenylTHF by enzymatic reduction is in the 
pathway for biosynthesis of serine and methio- 
nine. An enzyme has been purified from rabbit 
liver which carries out the following reaction: 
Imidazole acetic acid + l-pyrophosphoryl-5-phos- 

ATP 
phoryl ribose— ^imidazole acetic acid ribotide. 
This is the first demonstration in vitro of the 
mechanism of the riboside formation. The ribo- 
sides of histamine and imidazole acetic acid have 
been prepared by chemical synthesis. 

Sialic Acid. A highly sensitive and more specific 
method, using thiobarbituric acid, has been de- 
veloped for sialic acid. A histochemical method 
for stainino- sialic acid in tissues has also been 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



99 



developed. Using these methods several observa- 
tions have been made : species differences in sali- 
vary-gland content of sialic acid; a hormonal 
regulation of the amount of sialic acid in vaginal 
tissues (10-fold increase in pregnancy) ; an in- 
crease in thyroid cancer tissues ; large amounts of 
n-glycolyl and N-acetyl neuraminic in fish eggs. 
The sialic content of the thyroid gland parallels its 
thyroglobulin content and is diminished by thy- 
roid-stimulating hormone. 

Cholesterol Synthesis. The enzyme, mevalonic 
kinase, has been purified 100-fold from rabbit 
liver. Methods for identifying its products — 
phosphomevalonic and ADP — have been de- 
veloped. Requirements for the reaction include 
SH compounds, Mg, Mn, and phosphate. 

Gramicidin J. To elucidate the formation of this 
polypeptide antibiotic, Bacillus brevis has been 
grown with C 14 amino acids. Another compound 
closely related to this antibiotic has been syn- 
thesized enzymatically, and its structure is being 
determined. 

Enzyme Activity and Molecular Structure. 
It has been possible to degrade acetylated tryp- 
sinogen by pepsin and obtain fragments that re- 
tain a high degree of proteolytic activity. Such a 
fragment has been further degraded to 10 amino- 
acid residues by leucyl peptidase. By another pro- 
cedure it has been shown that, by treatment with 
bromsuccinimide, a differential destruction of the 
"specificity determining structure" and the "cat- 
alytic site" occurs. The localization of enzyme 
activity to specific sites on the protein molecule is 
of considerable importance. 

Sulfur Amino Acdds. Further knowledge of the 
metabolism of sulfur amino acids has been ob- 
tained from purified enzyme systems isolated from 
yeast. The reduction of methionine sulfoxide to 
methionine in the presence of TPNH was charac- 
terized. The enzyme system could be separated 
into three fractions, two of which catalyzed the 
nonspecific reduction of disulfides. In addition to 
these three enzymes, a new nonspecific disulfide 
reductase was isolated which reduced glutathione. 
The sulfur amino acid, felinine, is excreted in 
large amounts in the urine of feline species. By 
S 35 and C 14 labeling, it has been shown that a 



cholesterol precursor, mevalonic acid, is involved 
in its biosynthesis. 

Antibody Localization. By tagging antibodies 
with fluorescent dyes (Coons technique), a strep- 
tococcal antigen and the bacterial hyaluronidase 
have been followed in tissues after streptococcal 
infection in mice. By means of tagged antibodies 
to myosin, actin and sarcoplasmic proteins have 
been localized in the Purkinje cells of the conduct- 
ing bundle of the heart. 

Pharmacology of Iodate. A sensitive method 
has been devised for the detection of iodate in the 
urine. 

Laboratory of Biochemistry & Metabolism 

Carbohydrate Metabolism. The rate of degra- 
dation of glycogen by alkali under various condi- 
tions has been studied. Among the principal 
products of such degradations a number of mono- 
and polysaccharinic acids have been found. The 
most abundant of the monosaccharinic acids has 
tentatively been identified as iso-saccharinic acid. 
In other studies, the mechanism of action of a rat- 
liver transglucosylase has been studied. Its ac- 
tion does not involve phosphate compounds. The 
existence of a glucosyl enzyme intermediate is 
proposed. 

The finding that progesterone, testosterone, and 
androsterone stimulate the oxidation of D-ga- 
lactose in vitro has been further analyzed. The 
site of action of these hormones has been localized 
at the level of the uridine diphospho-galactose-4- 
epimerase reaction. This is one of the enzymatic 
steps by which galactose is converted to glucose 
and utilized in tissues. It happens that the 
epimerase enzyme requires DPN and is inhibited 
by DPNH. Accordingly, one mechanism by 
which progesterone could stimulate the epimerase 
reaction is by lowering the level of DPNH (and 
raising DPN) . It has now been found that some 
DPN-linked aldehyde dehydrogenase reactions 
in liver are inhibited by low concentrations of the 
hormone. Thus, by its effect on aldehyde dehy- 
drogenase, progesterone helps adjust the levels of 
the coenzymes DPN and DPNH which, in turn, 
affect a critical step in galactose utilization. 



100 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



A sensitive, highly specific, and comparatively 
simple assay for galactose-1-phosphate in erythro- 
cytes has been devised, that should be of value to 
physicians treating galactosemic patients. Treat- 
ment of three prepubertal galactosemic subjects 
with progesterone enables them to oxidize about 
10 percent of a tracer dose of D-galactose-1-C 1 * 
to C 14 2 . An improved purification of UDP 
galactose-4-epimerase (discussed above) from 
galactose-adapted yeast was devised and the puri- 
fied enzyme shown to contain tightly bound DPN. 
The yeast enzyme, like that from liver, failed to 
incorporate tritium into hexose nucleotide from 
either tritiated water or tritium-labeled DPNH. 
The role of DPN in the reaction thus remains to 
be clarified. 

Studies on inositol biosynthesis in the rat con- 
tinue to support the hypothesis that a six-carbon 
precursor (not glucuronic acid) is cyclized in 
some manner. This contrasts with the yeast sys- 
tem, where a two-carbon and a four-carbon unit 
combine to form this cyclic alcohol. Further, it 
has also been established that cleavage of inositol 
to glucuronic acid observed in rat-kidney extracts 
is not sufficiently reversible to account for inositol 
biosynthesis in the whole animal. 

Studies of the metabolism of insulin-I 131 by sur- 
viving rat liver have been carried out under a 
variety of conditions. The results of such studies 
have implicated the cell membrane as a possible 
determinant of the specificity of capture and deg- 
radation of the hormone by the intact organ. 
The results suggest that degradation of insulin- 
I 131 by intact liver may proceed in the following 
way : (1) binding of insulin by the cell membrane ; 
(2) transport of insulin to the site of insulinase 
activity; (3) degradation of insulin. 

A new pathway for the metabolism of uronic 
acids in bacteria, outlined in the last report, has 
been intensively studied and purification of all of 
the reactions achieved. This work is completed 
and has been submitted for publication. 

Preliminary studies on mucopolysaccharide for- 
mation have demonstrated the presence of an en- 
zymatic system in mouse-skin homogenates capable 
of synthesizing chonclroitin sulfate B, an iduronic 
acid containing polymer. Studies are under way 
to elucidate the biosynthetic mechanisms involved 
in the conversion of glucose to this new uronic 
acid. Concurrent studies in rat kidney have re- 



vealed that L-iduronic acid is readily metabolized. 
At least two enzymatic steps have been demon- 
strated, one of which has been purified about 2001- 
fold. This represents the first report of an idu- 
ronic acid metabolizing system. 

Continuing work on ascorbic-acid metabolism in 
collaboration with NHI has led to the identifica- 
tion of the two pentonic acids, L-lyxonic and L- 
xylonic acid, arising from diketogulonic acid. A 
second enzyme acting upon diketogulonic acid has 
recently been discovered which catalyzes the for- 
mation of 2-ketogulonic acid. The fate of this new 
intermediate, as well as its possible role in the 
mechanism of action of ascorbic acid, is being 
explored. 

An investigation has been undertaken of a new 
group of rare sugars, the 3,6 dideoxy hexoses, 
which are responsible for the immunological speci- 
ficity of the O antigen of Escherichia and Sal- 
monella. A rapid and sensitive assay technique 
has been developed for the determination of these 
compounds. Preliminary results indicate that 
these sugars are formed from glucose without re- 
arrangement or inversion of the carbon skeleton. 

Nucleic Acids : Structure and Metabolism. In- 
creasing emphasis in this field is being placed on 
the matter of secondary structure, the holding to- 
gether of polynucleotide strands by hydrogen 
bonds. Theories of genetic duplication and spe- 
cific nucleic acid synthesis depend on these hydro- 
gen-bond interactions in which only certain base 
pairs participate. 

A number of recent projects contribute to our 
understanding of such interactions. By means of 
infrared spectra in D 2 solution the tautomeric 
forms of the nucleotide components of nucleic 
acids are being studied. This question is funda- 
mental to the structure of nucleic acids because the 
possible modes of hydrogen bonding are deter- 
mined primarily by this structural feature of the 
component nucleotides. During the past year it 
has been found that polyinosinic acid exists in the 
keto form in aqueous solution and polycytidylic 
acid probably in the amino form, and that these 
tautomeric structures are maintained in the helical 
interaction product formed by mixing the poly- 
mers. 

A fundamental question is how large a poly- 
nucleotide has to be in order to interact and help 
to form a double helix. It has now been found 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



101 



that simple trinucleotides can line up in a chain 
and become attached by hydrogen bonds to a poly- 
mer, thus forming two chains bound together. 
For example, the trinucleotide pApApA will in- 
teract with the polymer, polyuridylic acid. 

The action of the bacterial enzyme, polynucleo- 
tide phosphorylase, is governed by the possibilities 
of hydrogen bond interaction. Thus, polymeriza- 
tion of ADP is inhibited by poly U and vice versa ; 
polymerization of CDP is inhibited by poly I and 
vice versa. This reflects the fact that poly A+ 
poly U, and poly I + poly C represent strong sec- 
ondary interactions. Every permutation has been 
tested, and the enzyme activity is strongly sup- 
pressed only by pairs where hydrogen bonding be- 
tween bases is possible. This could be the basis 
for specificity in RNA synthesis. 

An interesting finding from studies in collabo- 
ration with NIMH is that soluble RNA, so-called 
S-RNA, the acceptor for amino acids in protein 
synthesis, is phosphorylized very slowly by poly- 
nucleotide phosphorylase and reaction stops when 
20-30 percent of the soluble RNA has been con- 
verted to nucleoside diphosphates. Experiments 
now indicate that it is the nature of the secondary 
structure of the chains or hydrogen bonding that 
determines their resistance to degradation. Solu- 
ble RNA has a terminal phosphate endgroup at 
the beginning of the chain and the other end of 
the chain is called the "nucleoside end." The nu- 
cleoside end of the molecule consists exclusively of 
adenosine. Now it has been found that, in rabbit 
liver S-RNA, all of the chains begin with a 
guanylic-acid residue. A new nuclease has been 
discovered and purified from extracts of Azoto- 
bacter agiles, and its mechanism of action deline- 
ated. It promises to be very useful in studies of 
S-RNA. 

As stated above, S-RNA is widely believed to 
be involved in a scheme for protein biosynthesis. 
For example, in E. coli, S-RNA and a soluble en- 
zyme have been found necessary to incorporate 
certain amino acids into a particulate fraction. 
This system differs from previously described sys- 
tems in that it is not inhibited by chloramphenicol 
or lecithinase A. It also requires ATP, Mg ++ , 
inorganic phosphate, and a sulfhydryl-containing 
compound. 

Steroid Metabolism. The work on steroids has 
folloAved two separate courses. The first has dealt 



with the metabolism of these compounds and the 
second with their mechanism of action as hor- 
mones. 

As for their metabolism, considerable attention 
has been given to the reduction of the 4-5 double 
bond of hormonally active molecules. There are 
two sets of specific, TPNH-dependent enzymes 
for catalyzing this hydrogenation : the micro- 
somal 5a reductases, and the soluble 5/3 reductases. 
There are, therefore, for each steroid, two separate 
4-5 reductases. The microsomal enzymes respond 
to various physiological stimuli like thyroxin by 
increasing in activity. These two sets of similar 
proteins are being examined to determine if any 
biosynthetic or genetic relationship exists between 
them. 

The 11 -hydroxy lation of adrenal steroids, an 
important reaction in the biosynthesis of steroid 
hormones, and a good model reaction for all hy- 
droxylations, has also been investigated. Three 
enzymes, TPNH, and an unknown cofactor are 
required for this reaction. One form of the met- 
abolic disease, the adrenogenital syndrome, may 
be associated with loss of one of these enzymes. 

There have been several separate studies on the 
mechanism of steroid action : The first on steriods 
as inhibitors of DPNH-cytochrome c reductase. 
Here, the steroids act between flavoprotein and 
cytochrome c and their effect can be overcome by 
a-tocopherol or other lipids. Also it has been 
shown that TPN, produced from TPNH in the 
course of steroid double-bond reduction, can regu- 
late such processes as glucose-6-phosphate oxida- 
tion in tissues containing both the shunt pathway 
and the steroid reductases. 

In another study, intracellularly generated 
tritium labeled DPN, namely diphosphopyridine 
nucleotide-4-T, was used to evaluate the concept 
that hydroxysteroids are involved in liver trans- 
hydrogenase activity. To this end the several 
hydroxysteroids were tested for possible effect in 
catalyzing the approach to equilibrium of the 
DPN-DPNH/TPN-TPNH couple. No evidence 
came forth to suggest that, in liver cells, hydroxy- 
steroids functioned in a transhydrogenase 
capacity. 

Gene-Enzyme Relationships in Histidine Bio- 
synthesis. Work by Hartman (Johns Hopkins 
University) on mapping of the genes of histidine 



102 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



biosynthesis in Salmonella mutants has shown 
that they are all in a cluster on the chromosome. 
Biochemical analysis of these mutants has now 
revealed that the sequence of the histidine genes 
on the chromosome linkage map corresponds to 
the sequence of the enzymes they control in the bio- 
synthetic pathway. 

It has further been shown that histidine alone 
controls the rate of synthesis of the various en- 
zymes of its biosynthetic pathway ; if the histidine 
pool is increased there is repression, and vice 
versa. A major finding is that histidine affects 
the synthesis of each of the enzymes of the path- 
way to the same extent. It is as if histidine (or a 
derivative) had a specific affinity for the histidine 
section of the chromosome and could "turn off" 
these genes when the internal histidine concentra- 
tion rises. 

Role of Polyamines in the Neutralization of 
Bacteriophage DNA. It has been established 
that the cations putrescine ++ , spermidine +++ and 
Mg ++ neutralize the D1STA of T4 bacteriophage ob- 
tained from E. coli grown in minimal medium. 
The cations in T4 phage have been shown to be a 
function of both the composition of the pool of 
cations in the host bacterium at the time of phage 
assembly and the affinity of each species of cation 
for the phage nucleic acid. 

Enzyme Induction, y -Hydroxy butyric acid was 
found to induce the formation of y-hydroxybu- 
tyric acid dehydrogenase at low inducer concen- 
trations. Higher concentrations of y-hydroxybu- 
tyric acid were highly effective inducers, not only 
of y -hydroxy butyric acid dehydrogenase, but also 
of /3-hydroxypropionic acid dehydrogenase. The 
question whether the same genetic unit (cistron) 
contains the information necessary for the syn- 
thesis of a protein subunit which may be an in- 
tegral part of both enzymes was studied. There 
was no evidence of shared genetic information. 

Enzymatic Utilization of Model Compounds. 
Studies on the biosynthesis of y-aminobutyrate 
from pyrrolidine and putrescine and the subse- 
quent utilization of this compound have been 
studied at the enzyme level. Of particular interest 
has been the study of the kinetics of one of the 
reactions involved in y-aminobutyrate utilization, 



namely, its transamination with a-ketoglutarate, 
forming succinic semialdehyde and glutamate. 
The data had suggested that transamination 
occurred by way of a series of binary com- 
plexes of enzyme and each substrate. Further 
support for this concept has been obtained by the 
dissection of the transamination into two ex- 
change reactions. 

In a study of a novel aldehyde dehydrogenase 
oxidizing malonic semialdehyde, both DPN and 
CoA were found to be involved, resulting in the 
direct formation of C0 2 and acetyl-CoA. Free 
malonyl-CoA was not formed. 

Coenzyme Studies. Synthesis of thiamine has 
now been shown to involve the initial formation of 
thiamine monophosphate rather than the free vita- 
min, as claimed by others. The synthesis involves 
three enzymatic steps: The phosphorylation of 
the pyrimidine to the corresponding pyrimidine 
pyrophosphate; the phosphorylation of the thi- 
azole to thiazole monophosphate; the condensa- 
tion of these derivatives to form thiamine mono- 
phosphate with the elimination of pyrophosphoric 
acid. 

Laboratory of Pathology and 
Histochemistry 

Hematologic and Genetic Studies. Continued 
investigation of factors influencing erythropoiesis 
has revealed a correlation of plasma erythro- 
poietine with the grade of marrow cellularity and 
an accelerating effect of erythropoietine on red- 
cell maturation. Attempts to assay erythro- 
poietine by use of short-term tissue cultures have 
not been successful. Studies using tritiated thy- 
midine have shown that erythropoietic cells do not 
enter mitosis in vitro. A second regulator of 
erythropoiesis has been postulated to account for 
some of the results obtained in recent experiments. 
Studies on the genetic control of hemoglobin 
structure have been continued. A molecule of 
hemoglobin, which is composed of a pair of alpha 
chains and a pair of beta chains, dissociates re- 
versibly in acid into the two unlike pairs. These 
pairs are exchanged between molecules when a 
mixture of two different hemoglobins is dis- 
sociated and recombined. With use of this tech- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



103 



nique it has been shown that the two types of 
chains, alpha and beta, are controlled by different 
genetic loci. The reactivity of hemoglobin SH 
groups toward each other can be greatly increased 
by combination with nitrobenzene. Tryptic diges- 
tion and paper electrophoresis followed by paper 
chromatography has shown that fraction 23 of 
hemoglobin I contains tryptophan whereas this 
fraction of normal hemoglobin does not. Search 
for other amino-acid differences between these 
hemoglobins is underway. 

Studies have been instituted on human karyo- 
types in relation to localization of specific genes. 
Sex-chromosomal variations in clinical and latent 
hermaphroditism and chromosomal variations in 
neoplasia and in male meiosis are also being in- 
vestigated. 

Experimental Arthritis. Studies of the experi- 
mental arthritis induced in rats by Streptobacillus 
moniliformis have demonstrated that a focal 
osteomyelitis proceeds to periostitis and synovitis 
by local invasion and thence to joint involvement. 
The organism has proved infectious for mice, and 
is progressively less lethal after intravenous, in- 
traperitoneal, and subcutaneous inoculation. 
Grossly evident joint lesions appear in as little as 
1 day and persist for as long as 3 months. 

Blood from infected rats shows a positive Ben- 
tonite flocculation test and sensitized sheep-cell 
hemagglutination reaction similar to that seen in 
human rheumatoid arthritis. These reactivities 
have been produced in sera of rabbits immunized 
with formaldehyde-killed cultures of infective 
strains of S. moniliformis. Immunochemical 
studies indicate a small molecular size for the 
active fraction, contrasting with the large molecu- 
lar size of the human rheumatoid serum factor. 
The organism apparently must be grown on 
human ascites fluid to be antigenic, but ascites 
fluid itself does not contain the antigen in 
question. 

Studies on degenerative joint disease of mice 
have continued. Genetically, susceptibility to os- 
teoarthritis behaves as a recessive; studies de- 
signed to determine the number of genes involved 
will be completed shortly. At the time of this 
report, the findings suggest that there is a single 
gene factor. The sources of different suscepti- 
bility of various inbred strains of mice were 
found not to reside in the differences in rates of 



skeletal aging (epiphyseal development), thyroid 
function, obesity, or response to high fat diets. 

The swellings of the paws of rats following 
injection of Freund adjuvants were demonstrated 
as not representing arthritis but a periarthritis. 
Evidence was presented that such swellings arose 
from dissemination of adjuvant from the depot 
site to the periarticular tissue. 

Two interesting, genetically determined, spon- 
taneous diseases of mice have been observed for 
the first time during the course of these studies: 

(1) a polydipsia that leads to lethal hydroneph- 
rosis in males of strain STR/N. (The findings 
suggest that there is a primary disturbance of 
thirst mechanism to account for the syndrome), 

(2) a pelvic inflammatory disease in male STR/I 
N mice that is related to the seminal function of 
this strain. 

A paper on the pathogenesis of ochronotic ar- 
thropathy, based on anatomic, electromicroscopic, 
and in vitro chemical studies of the combination 
of homogentisic acid and articular cartilage is in 
preparation. Other reports on the pathology of 
human rheumatic diseases published during the 
last year have dealt with joint involvement in 
sarcoidosis and the genesis of rupture of extensor 
tendons at the wrist in rheumatoid arthritis. 

Infectious Processes. A staphylococcal endo- 
carditis was produced experimentally by an intra- 
venous injection of staphylococci in dogs rendered 
susceptible by prior surgical induction of aortic 
insufficiency. Endocarditis could be prevented 
by penicillin given within 8 hours after injecting 
the bacteria. If treatment was delayed 24 hours 
or longer, relapses often occurred after cessation 
of treatment. The endocarditis was arrested by 
treatment of relapses, but some animals died of 
acute heart failure due to valvular deformities 
and insufficiency. After delayed therapy, nearly 
all dogs developed a diffuse proliferative glomer- 
ulonephritis which resisted therapy and occa- 
sionally showed evidence of developing into 
a chronic glomerulonephritis. These findings 
support the concept, recently questioned, that a 
chronic glumerulonephritis is a sequel to an acute 
nephritis. This experimental method should 
prove a useful tool in studying staphylococcal in- 
fections resistant to antibiotic therapy and in the 
study of glomerulonephritis. 



104 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The pathologic process engendered by inocula- 
tion of a virulent strain of Trichomonas vaginalis 
into mice by the intraperitoneal route has been 
characterized but the mechanism of the resulting 
liver-cell destruction remains to be elucidated. 

Stress and Endocrine Effects. Repeated ex- 
posure of dogs to a 30,000- foot-simulated altitude 
resulted in the development of nonlipid arterio- 
sclerotic changes in the aorta and occasionally in 
the coronary arteries in a substantial portion of 
the dogs. This new experimental method of pro- 
ducing arteriosclerosis supports the concept that 
anoxemia may be a cause of arteriosclerosis. 

Studies conducted in collaboration with NHI, 
showed that adrenergic blocking agents prevent or 
diminish fat deposition in the liver occurring 
after the administration of CC1 4 , ethionine or eth- 
anol. The adrenergic blocking agent, phenoxj'- 
benzamine, also inhibits a rise in serum lactic de- 
hydrogenase as well as a rise in serum transamin- 
ases and fatty myocardial changes reported pre- 
viously in dogs given large doses of norepineph- 
rine of epinephrine. However, phenoxybenza- 
mine or reserpine does not prevent a similar rise 
in serum-enzyme levels and the fatty changes bor- 
dering myocardial infarcts produced in dogs by 
coronary ligation. Since reserpine depletes the 
myocardium of norepinephrine, these findings do 
not support the hypothesis that ventricular tachy- 
cardia is due to epinephrine and norepinephrine, 
liberated from the infarcted myocardium. 

Histochemical Studies. A combined critical re- 
view and an original study of the histochemical 
reactions of human gastrointestinal carcinoid tu- 
mors has been completed. The routine use of fer- 
ric ferricyanide and a stable diazotate is recom- 
mended to get more data on functional activity 
of these tumors. A new metal chelation reaction 
with enterochromaffin is perhaps suggestive of an 
<?-diphenol structure, or o-secondary aminophenol. 
Studies have been continued on the localization 
of oxidative or dehydrogenase enzymes using 
tetrazoles as histochemical indicators. The pres- 
ence of both TPN- and DPN-linked 17^-estradiol 
dehydrogenases in placenta has been demonstrated 
with distinctly different topochemical distribu- 
tion patterns. 



Studies with NIDR on aminopeptidases in hu- 
man tumors have been extended. Some epithelial 
tumor types possess this peptidase activity in the 
tumor cells themselves. More numerous epithelial 
tumors exhibit proteolytic activity in adjacent 
stroma, independent of proliferation of stroma or 
inflammation. A third category of tumors includ- 
ing sarcomata invade without evident proteolytic 
activity. 

A chromogenic substrate specific for trypsin 
was synthesized and this type of activity has been 
demonstrated in mast cells, though not in tumor 
stroma. The diazotisation procedure recently de- 
veloped in this laboratory for demonstration of 
tyrosine in protein has been used to show high 
tyrosine concentrations in hypophyseal alpha cells. 

Mucins and chemically related substances in 
rodents have been studied by means of a variety 
of histochemical procedures. Methylation-de- 
methylation sequence procedures have been used 
for the distinction of sulfate from carboxyl acid 
mucins, the former being methylated and con- 
verted to neutral mucins, the latter being esteri- 
fied, saponified, and restored by acid to the 
original status. S 35 4 incorporation has been 
compared with alcian-blue staining and thiazin 
uptake at pH 1.5 and 3.0, and aldehyde fuchsin 
staining. Sulfated mucins stain at the lower pH, 
some are nonreactive to alcian blue and to periodic 
acid Schiff stain. Sialic acid mucins generally do 
not stain with thiazins at pH 1.5 but do at pH 3. 
They take alcian blue and are digestible with V. 
cholerae and CI. perfringens sialidases with libera- 
tion of recoverable sialic acid. Topographic dis- 
tribution studies indicate a good deal of specific 
localization of the several types of mucopolysac- 
charides. 

Studies of the histochemical reactions of neu- 
romelanin in man have allowed the identification 
of this material in substantia nigra, locus caeru- 
leus, and nucleus dorsalis vagi of Macaca mulata. 
The eosinophilic granules which are mingled with 
neuromelanin granules in man owe their eosino- 
philia probably to e-amino groups of lysine and 
hydroxylysine, and seem to include a phenolic 
substance other than tyrosine, histidine or 
tryptophan. 

Studies have been made on the rat preputial 
gland, assessing response of the two separate de- 
monstrable secretion products to hormonal stimuli. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



105 



The histochemistry of the gland secretion prod- 
ucts has been reported. 

Eenal Architecture. Thru collaboration with 
NCI, electron microscopy has been applied to the 
study of the counter-current vascular bundles of 
the renal papilla. The structure of these vessels 
proved to be essentially identical to that of the 
vessels in the well-known retia mirdbilia of the 
toadfish swimbladder. Continuous basement 
membranes are displayed in both ascending and 
descending vasculature. 

Glutaminase has been localized to the proximal 
convoluted segment and the distal straight seg- 
ment of the rat nephron. The distribution and 
amount of glutaminase found in the distal tubule 
cannot readily account for the ammonia excretion 
in the renal papilla of the rat, and therefore some 
other mechanism for this excretion must be found. 

Chlorophenol red is apparently excreted in the 
proximal convoluted segment, and is resorbed, at 
least partially, in the proximal, straight segment. 
Whether the resorbed dye is returned to the venous 
circulation has not been determined. This might 
account for incomplete clearance. 

Germ-Free Animals. Studies in germ-free ani- 
mals have been directed to the pathogenesis of 
liver cirrhosis. Choline-deficiency liver cirrhosis 
in rats develops quite typically and within the 
usual time range. It has been alleged by other 
workers that the lesion is attributable at least in 
part to intercurrent infectious processes. 

Human Pathology. Studies of the geographical 
and racial differences in human pathology have 
been continued. Autopsy material collected in 
Japan by the Atomic Bomb Casualty Commission 
is being studied for degenerative cardiovascular 
diseases among the Japanese of Hiroshima and 
will be compared with similar published data for 
American and European peoples. Along with 
NINDB, this laboratory also is participating in 
a pathologic and experimental study of the neuro- 
logic disorder in man and animals produced by 
the eating of fish caught in Minamata Bay in 
Japan. Japanese workers have reported that this 
disease may be an organic mercurial poisoning 
from industrial wastes. From pathologic mate- 
rial obtained through the diagnostic services pro- 



vided to Indian hospitals, this laboratory has 
noted a dietary hemosiderosis and an obscure 
hepatic alteration in American Indians. A patho- 
logic study of sarcoidosis in American Indians of 
Oklahoma is being supplemented by cooperative 
epidemiologic studies conducted by the University 
of Oklahoma Medical School. 

An outbreak of pneumocystic carinii disease in 
a Korean orphanage has demonstrated a non- 
European focus of this disease. Promising re- 
sults have been obtained by induction of this dis- 
ease in rats. 

The histopathology preparation laboratory pro- 
vides services needed by two sections of LPH, 
and continues to give aid and consultation on 
technical problems to scientists in many other 
laboratories. During the past year this laboratory 
prepared a total of 49,181 sections : 16,555 routine, 
18,254 special stains, and 14,372 spare slides; 
from 2,193 surgical specimens, 103 human autop- 
sies and 5,484 animals. Animals were derived as 
follows: 3,346 from DBS, 1,137 from NIAMD 
and 1,001 from other Institutes. This total does 
not include the large number (approximately 
19,000) of sections prepared by a large variety of 
procedures within the Histochemistry Section. 

Laboratory of Physical Biology 

The Laboratory of Physical Biology comprises 
a broad spectrum of fundamental biological and 
related research, much of it independently execut- 
ed but invariably arising from interrelated prob- 
lems which attract the interest and curiosity of 
many scientific disciplines. The approaches to 
the analysis and elucidation of the diverse factors 
impinging on living systems vary from the investi- 
gation of the role of structure in function, 
from molecular aggregation to histology, to the 
analysis of the energetics of the interaction of en- 
vironment and organisms. The past year has seen 
the addition of staff in biophysics particularly in 
the section of photobiology and, with its cleavage, 
to form a new section on bionucleonics to attack 
separately the problems of high-energy radiation 
interaction with biological systems. Problems of 
space have become increasingly acute and, despite 
cooperation of sister laboratories off campus in 
housing some of our staff, we look forward to re- 



106 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



lease of space indirectly through the current build- 
ing program of other Institutes. 

In view of the very diverse nature of investiga- 
tions coming under the planned experimentation 
of this laboratory, the following segments have 
been summarized somewhat arbitrarily and no sig- 
nificance is to be attached to the order of presenta- 
tion. In no case is it possible to include all the 
results of the year's work or to give individual 
credit for particular findings. The bibliography 
of the laboratory staff and the directory of the 
laboratory will show how ably this heterogeneous 
staff works both cooperatively and independently. 

In the area of photobiology, studies on basic 
photosynthesis have shown that the organization 
of pigment-protein molecules into a functional 
network may well explain the extraordinary ef- 
fectiveness of this "machine." It was shown that 
a partially reversible change in the bleaching of 
chlorophyll is dependent on oxygen and that the 
site of this action is related to the lamellar chloro- 
plast structure. If one digests lipoid from the 
chloroplast, the still intact layers are free to sepa- 
rate in fan-like fashion. On the other hand, pro- 
tein digestion causes the layers themselves to col- 
lapse. Other studies relating structure to func- 
tion in this area show that anomalies in the scat- 
tering of radiation by pigments account for only 
a small fraction of the wavelength change describ- 
ing the difference between free pigment and the 
same material in vivo — an indication that this 
wavelength shift may relate to the extent of 
macromolecular organization of structure. Find- 
ings on the structure of porphyrins are particular- 
ly interesting in that specific stoichiometric bind- 
ing is exhibited by copper porphyrin to bovine 
serum albumin whereas there is a contrasting lack 
of such binding in /Mactoglobulin. In other 
physical examinations it is evident that specific 
electrochemical properties characterize such bio- 
logically functional structures — in particular, the 
demonstration by nuclear magnetic resonance that 
the conjugated porphyrin molecule carries "ring 
currents" producing local magnetic fields within 
the molecular structure due to the induced circula- 
tion of the ir electrons. 

In the allied field of the basic mechanism of 
vision the way in which energy is absorbed and 
transferred is under study and in the coopera- 



tive work with the Naval Medical Research Insti- 
tute good agreement with theory has been shown 
on the minimum electrical current that a photo 
receptor must produce (about 1,000 charges/ 
photon) to convey information to the brain. This 
value has been measured as 750 electrical charges 
per incident photon in photo-receptors of the 
squid. The underlying physicochemical bases for 
these biological functions are of direct interest to 
chemists who relate such properties to triplet and 
metastable states of the molecules and their ag- 
gregates. Thus, pigments having photodynamic 
properties are studied for clues to mechanism of 
energy economy. For example, a red pigment 
from a fungus has been found to exhibit an usual 
pattern of conjugation and to have photodynamic 
properties, in particular, a destructive photo-oxi- 
dation which may be able to furnish energy to use- 
ful endothermic reactions in the organisms. 

It has been shown in this section that the con- 
trol of energy through wave-length specificity for 
unit molecular action and resonance phenomena is 
of great importance to the economical and specific 
production of certain essential chemical and bio- 
logical substances. Thus, only ultraviolet light 
will produce the conversion of 7-dehydro-choles- 
terol into vitamin D 2 (calciferol), cause erythema, 
bactericidal action, and mutations in various or- 
ganisms. The section has carried on quantum 
measurements of ergosterol transformation in a 
variety of monochromatic irradiations and con- 
ducted flash photolysis studies to reduce the inci- 
dence of thermal changes during irradiations. An 
extensive reexamination of monochromatic irra- 
diation data on the reaction of precalciferal and 
tachysterol which tends to show through flash 
photolysis that a dark reaction exists in this direc- 
tion following irradiation. 

In extension of these interests the section is 
looking into the problem of genetic changes pro- 
duced by resonant energy mechanisms for spec- 
ificity to wavelength as contrasted to the high- 
energy effects of radiation. Current studies are 
of a cooperative nature on chromosomal altera- 
tions as related to lethality of dose. 

At the high-energy level, studies are just be- 
ginning to be set up for elucidating the effects of 
neutrons and X-radiation in nonlethal dosages 
partly by study of molecular interactions and 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



107 



partly by cytological changes which can be ob- 
served. Staffing and equipping of this endeavor 
have been major problems. 

The physicochemical study of living systems 
may require the examination of basic nonliving 
models which exhibit and perhaps elucidate the 
complex membrane-system integral with biologi- 
cal function. In the section on macromolecular 
biophysics this type of investigation has laid a 
broad groundwork leading to the rational inter- 
pretation and prediction of properties of ion trans- 
port through various types of membranes with 
definable characteristics. Thus, in the current 
work, porous membranes of highly specific ion- 
transfer characteristics have been prepared repro- 
ducibly and their mechanisms analyzed and re- 
lated to theory. This has been extended to oil 
membranes of similar characteristics as a step 
toward the more complex living-tissue interfaces. 
The behavior of various components of these mem- 
brane-solvent-ion systems has been shown to in- 
clude the concept of membrane hydrolysis which 
appears to act against theoretically derived esti- 
mates of membrane potentials at low electrolyte 
concentrations. Further studies demonstrating 
the inability of the "Donnan equilibrium" concept 
adequately to describe the conditions obtaining in 
conventional aqueous concentration cells are being 
carried on and from a theoretical analysis of the 
problem it can be seen that such preparations must 
be viewed as dynamic "two-ionic" cells to which 
the theory of the dynamic aspects of polyionic po- 
tentials recently developed here must be applied. 

Studies in molecular biophysics have proceeded 
also to the analysis of the structure of protein 
crystals by electron microscopy of very high reso- 
lution and the use of a minimum assumption model 
to reproduce the observed phenomena. The crys- 
tal structure of Rothamsted tobacco-necrosis pro- 
tein has been determined in this manner and is in 
close agreement with the crystal structure found 
using X-rays. The photography of molecular 
separations in crystals of organic compounds of 
molecular weights of about 500 to 700 has been 
continued and it has been determined that the 
image obtained is not a direct one but an inter- 
ference pattern produced by phase changes in the 
electron waves passing between and through the 
planes of properly oriented crystals. It is of sig- 
nificance to the analysis that the images appear 



not in the principle focus but above and below it 
in a predictable pattern. Further development 
of microspot X-ray microscopy to photograph 
diffraction patterns using long wavelength X-rays 
(up to 10 A) has resulted in achieving increased 
dispersions on the recording plate over those pos- 
sible by former and currently available devices 
and permitting the easy determination of large 
molecular plane spacings in crystals. The work 
of the section has made it possible to image more 
accurately and to interpret the fine structure of 
biological and organic molecular structures ap- 
pearing in high-resolution electron micrographs. 

The section on physical biochemistry is devoted 
to an intensive analysis of the various manifesta- 
tions of the relationships between structure and 
function at the molecular, cellular, and organ level 
in biology. Its staff continues to analyze the 
mechanical, chemical, and energetic factors in the 
special protein complexes which account for the 
unique behavior of muscle, of blood clotting, and 
of a number of allied systems in which the inter- 
conversion of energy and structure takes place 
through highly specialized properties of discrete 
parts of oftentimes huge molecular aggregates. 
The concepts of enzyme-substrate relationships 
are met in this area and have proved useful in 
analysis, characterization, and prediction of ac- 
tion. A continuing study of the manner of poly- 
merization of muscle proteins has been carried on 
as well as related studies on the behavior of fibrino- 
gen and the essential alterations in producing 
netted structures of special attributes which 
produce the gross effects. 

In X-ray studies of glycerol-treated muscle fiber 
it was shown that a-keratin patterns disappeared 
in contractions induced by ATP, indicating a type 
of "melting" of the ordered filaments which leads 
to the assignment of two roles for ATP : one, the 
initiation of contraction in which no appreciable 
energy is lost by ATP and, secondly, the furnish- 
ing of energy ultimately by acting as the restora- 
tive agent to bring the contracted structure to its 
original oriented state. Other studies of muscle- 
fiber relaxations were carried on through the use 
of polyphosphate in the presence of ATP. Studies 
of the detailed structure of myosin are under way 
as a necessary prerequisite to the understanding 
of its function in the process of contraction. Com- 
parative myosin studies have been made from 



108 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



widely different species as well as from various 
tissues of the same organism and, in the case of 
the mammalian heart, it has been shown that the 
conduction bundle tissue which functionally is 
similar to nerve tissue still comprises protein con- 
stituents which are characteristic of muscle, thus 
supporting the inference of common physicochem- 
ical bases for action tissue. 

Studies on actin characterization indicate a 
great similarity to myosin and support the hy- 
pothesis that actin is a part of the larger myosin 
entity. The process of the transformation of 
globular actin to fibrous actin has been followed 
by optical rotation studies and microcalorimetric 
measurements and shown to be due to a gain in 
order with energy derived from ATP. 

Structural studies on other proteins including 
salmine and various micelle-forming substances 
such as soaps and detergents are being carried on 
to determine the role of specific structure and or- 
der of structural components with the hypothesis 
that such relationships may determine specific ca- 
pacities especially with regard to the transmission 
of genetic information content. The relations of 
sulfhydryl content of proteins are also under study 
with special reference to the correlation of reversi- 
ble dissociation of hemoglobin and the Bohr effect 
as functions of pH and sulfhydryl changes. Stud- 
ies on the metabolism of collagen in various 
conditions are continuing with peripheral studies 
of substances appearing as metabolites and detect- 
able by chromatographic procedures. 

In the correlated studies on proteins in this sec- 
tion the enzymatic nature of the processes by 
which proteins manifest their biological functions 
is under intensive investigation. Through success- 
ful purification of thrombin it has been made pos- 
sible to study their kinetic-molecular properties. 
It has been shown that the Laki-Lorand factor 
liberates a single peptide from fibrinogen through 
the action of thrombin This specificity of throm- 
bin was studied by the preparation of peptides of 
arginine which demonstrate the different effects 
of various structures on the splitting role of the 
reaction. An esterase activity of thrombin was 
demonstrated which seems to be exhibited toward 
all basic amino acids. 

Carboxypeptidase A has been intensively studied 
with the finding that binding cobalt on the zinc- 
bound enzyme enhances its activity. Carboxypep- 



tidase B has been isolated in a highly purified form 
from pig pancreas and seems to be a zinc metallo- 
protein strikingly similar to the A form although 
its specificity is markedly different. Trypsin has 
been shown to bind a second site of DFP 32 . A pep- 
tide of 19 amino acids is isolable which is different 
from that released from the first site of binding at 
the site of inactivation. A complete structural 
characterization is being undertaken. A similar 
definition of sequence has already been carried out 
for the peptide A liberated from fibrinogen during 
the clotting process. New studies on enzyme com- 
plexes have been initiated and attempts to 
fragment these complexes with preservation of ac- 
tivities is underway. Associated studies in im- 
munochemistry have been carried forward with 
the observation that serological reactions of rats 
and rabbits to killed Streptobacillus monoliformis 
are largely due to immunization with gamma glob- 
ulin from human ascites fluid as corroborated by 
immunization with cultured organisms in media 
containing human ascites fluid. 

In the section on physiology, which supports a 
wide range of function studies, the work on non- 
mammalian forms has been centered about several 
different phyla. The cyclic gonadal differentia- 
tion and its controlling factors in freshwater hy- 
dra are being studied in order that this useful 
preparation may yield basic information on cellu- 
lar responses and perhaps ultimately be available 
as a biological index of the effect of various envi- 
ronmental factors, including various forms of 
radiation on biological rhythms. Numerous pos- 
sible factors such as C0 2 concentration and popu- 
lation density have been shown to be noncritical 
in their effect. 

A further section of study is devoted to insect 
metabolism and constitutes the continuing core of 
interest with regard to respiratory phenomena in 
metabolic cycles of unusual interest. The finding 
of high citrate levels in the blood of five species of 
insects examined to date led to the hypothesis that 
this is a biochemical peculiarity of insects but it 
has been shown that it is not the result of a block- 
age of later stages of the TCA cycle since the req- 
uisite enzymes are demonstrable. The in vitro 
oxidation of citrate, alphaketoglutarate, malate, 
fumarate, and pyruvate has also been demon- 
strated by insect mitochondria. Further studies 
will follow organic-acid metabolism during vari- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



109 



ous stages of development and. a study of amino- 
acid hormones and protein synthesis using lysine 
C 14 . 

The study of the relation of oxygen tension and 
temperature to respiration of fly larvae and pupae 
has been completed and is being compared to the 
adult stage. It has been shown that larval respira- 
tion is not limited by physical dimensions as 
contrasted to limitation in pupae and that tem- 
perature dependence of larval respiration shows a 
discontinuity between 10-15° C. Central-nervous- 
system control or its absence does not affect oxygen 
uptake over the range of (Ml C. in day-old flies. 

Other studies on trauma indicate a difference 
between diapausing types of moth pupae and those 
which have no diapause. The study of the trig- 
gering of the flash of fireflies was continued this 
year with the finding that the photogenic tissue 
exhibits action potentials preceding flashes, that 
the latent period is separable into two compo- 
nents of which the first and longer one can be by- 
passed by intense stimuli, and that a variety of 
agents, including eserine and veratrine, can dis- 
rupt the lantern's coordinating mechanism to cause 
asynchronous activity. 

Proceeding to physiological studies of higher 
forms, the section's work on hypoxia has continued 
with the examination of altitude tolerance in 
chickens, rats, rabbits, and dogs. While the work 
is of a comparative character, the particular spe- 
cies used have certain differences in reaction which 
lend themselves to the elucidation of basic factors. 
As in previous years the studies show hemato- 
poietic stimulation in mammals, but the interest of 
the staff is held by results indicating that body 
temperature in rats affects altitude tolerance and 
that such subtle factors as restraint of the animals 
has a marked effect on body-temperature mainte- 
nance. It was also shown that dogs with experi- 
mental aortic insufficiency, mitral insufficiency, 
have an unsuspected and remarkably high altitude 
tolerance despite these drastic cardiac disabilities. 
Further findings of lipid atherosclerotic plaques 
in the hearts and aortas of dogs exposed to altitude 
suggest that hypoxia may play an important role 
in the etiology of this disease. 

Studies of the mechanism of circulatory re- 
action of sensitive species to synthetic macromole- 
cules have been continued in the section and deal 
with dextran and polyvinylpyrrolidone effects in 



rats and dogs, respectively. It was found that 
conscious rats showed no reaction to dextran if a 
proper combination and dosage of antihistamine 
and antiserotonin drugs were administered in con- 
firmation of independent results elsewhere on 
anesthetized rats. Since H. pertussis-moc\x\a,t\on 
of rats increases their susceptibility to exogenous 
histamine and serotonin, and since dextran is a 
histamine-and serotonin-releasing agent in rats, 
H. pertussis was given to rats subsequently given 
dextran. Susceptibility remained unchanged but 
counts of mast cells (the most important source of 
histamine and 5-hydroxytryptamine in rats) de- 
creased in the target areas of dextran sensitivity 
after inoculation and before reaction tests, thus 
tending to show that the presence of mast cells is 
not prerequisite for the reactions. Other studies 
on the effect of insulin on dextran reactions, for 
which other investigators have reported both en- 
hancing and mitigating roles, show that it acts 
in either manner depending on the route of ad- 
ministration and the dose of both dextran and 
insulin. Dextran at low closes tends to induce 
fatal convulsions in rats receiving nonconvulsive 
doses of insulin. There is evidence in dye diffu- 
sion and signs of thirst for an effect of insulin 
through its known action to increase cellular per- 
meability to carbohydrates. 

Finally, the section has continued to devote 
some study to environmental factors affecting 
pulmonary ventilation in human subjects. It was 
shown that time-lag between rise in alveolar pres- 
sure and the resultant mouth airflow is correlated 
with frequency of the breath and the effort 
exerted. The enhancement of this effect with 
denser gas mixtures than air and its mitigation 
with light mixtures has been demonstrated in 
accord with theory. 

Laboratory of Chemistry 

Analgesics. With a view to developing a new 
approach to compounds of the benzomorphan 
type, investigations on the dihydropiperidine sys- 
tems have been initiated, directed towards selec- 
tive alkylation at the 2 and 3 positions. Thus, 
research has continued in this family of neuro- 
pharmacologic agents (of which phenazocine is 
the presently most notable example) and includes 



110 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



further study of the physical dependence capacity 
of several of the more active members (particu- 
larly optical isomers). A significant number of 
these, ranging in potency from 5 times that of 
morphine to twice that of codeine, appears to be 
ineffective in suppressing morphine abstinence in 
the monkey at relatively high (subtoxic) doses. 

In addition, a new series of benzomorphans (9- 
hydroxy derivatives, analogs of the potent oxy- 
morphone and oxycodone derived from thebaine) 
have been synthesized and found to have interest- 
ing pharmacological properties. It is of special 
interest that the synthetic sequence we have de- 
vised for these compounds provides us with steric 
control in the formation of the carbinol grouping. 
Thus it is possible to obtain in excellent yield the 
racemate corresponding in configuration to oxy- 
codone (OH and iminoethano groups tis) or to 
completely reverse this stereochemistry to obtain 
the opposite racemate and a configuration not 
known in the morphine group. The charge on the 
nitrogen atom appears to be the major directing 
influence, although steric hindrance may also be 
a consideration. 

Research is being pursued toward devising a 
phenazocine-like structure containing an ether 
bridge characteristic of morphine, and toward 
simple neurotropic agents based on acetyl choline. 

As for the present status of phenazocine, the 
Smith, Kline & French Laboratories have applied 
for and expect to obtain, shortly, a new drug li- 
cense in the hope of putting phenazocine on the 
market by December 1. Five other U.S. firms 
have been licensed to manufacture phenazocine 
(at least four are going ahead with pilot-plant 
synthesis) , but foreign rights will not be exercised 
by DHEW. A publication for world-wide dis- 
semination of all pertinent data has been recom- 
mended. Finally, completed, short-term addic- 
tion studies at the Addiction Research Center, 
Lexington, Ky., and scattered clinical observa- 
tions indicate less addiction potential for phena- 
zocine than for morphine. 

Carbohydrates. The chemical synthesis of 2-de- 
oxy-D-ribofuranose nucleosides starting from the 
sugar itself was achieved for the first time. A 
new pathway to the synthesis of the component 
parts of deoxyribonucleic acid, the central sub- 



stance in the mechanism of heredity, was thus 
made available 

2-Deoxy-D-ribofuranose 1-phosphate, the inter- 
mediate in the enzymatic synthesis and scission of 
deoxyribonucleic acid, was synthesized by chemi- 
cal means for the first time. 

A wholly new pathway to the synthesis of nu- 
cleosides, involving 1-thiosugars was discovered. 

A simple method for the preparation of 3- 
deoxy-D-glucose and 3-deoxy-D-mannose from 2- 
deoxy-D-ribose was developed. These 3-deoxy- 
sugars are of some importance in current studies 
on the mechanism of insulin action. 

An eight-carbon sugar, D-glycero-D-manno- 
octulose, has been discovered in the avocado and 
in a Sedum species. This is the first known occur- 
rence of an octose in nature. 

Metabolites. In connection with fundamental 
studies in peptides and proteins, selective cleavage 
reactions have been elaborated with tryptophyl, 
tyrosyl, and methionine peptide bonds. Such 
studies obviate the great need for rapid methods 
for the establishment of the primary structures of 
proteins. They form part of a concerted effort 
to put available sequence data of amino acid com- 
binations on punched cards and subject them to 
information analysis. 

Thus, one out of more than 560 peptide bonds 
in human serum albumin (molecular weight 
around 70,000) was selectively split by a purely 
chemical method. Bovine and avian albumins 
were subjected to similar cleavages and revealed 
species differences in the amino-acid sequence fol- 
lowing the two and three molecules of tryptophan 
per molecule of protein. This new chemical 
method allows the rapid classification of albumins 
and is a simple test for the discovery of genetic 
and species changes in proteins. 

The use of this chemical cleavage method has 
allowed new insights into the structure of the pro- 
tein of tobacco mosaic virus and revealed the 
presence of three tryptophan units in the molecule 
(molecular weight 18,200), followed by alanine, 
lysine, and threonine. The try-lys and try-ala 
bonds show normal reactivity and cleavage, the 
new try-threo bond is unreactive in the large A- 
protein and reactive in the new C-terminal peptide 
containing 16 amino acids. This lack of reactiv- 
ity is attributed to secondary and tertiary effects. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



111 



The application of the N-bromosuccinimide 
cleavage to the vasoconstrictor peptide angiotensin 
has led to cleavage at the tyrosyl-peptide bond 
preceding the special amino acid which changes 
from species to species. 

The cyclic antibiotic gramicidin A, containing 
>40 percent tryptophan (prepared in a pure form 
in collaboration with the Rockefeller Institute) , is 
resistant to enzymatic hydrolysis and not suitable 
for conventional chemical cleavage; it has now 
been selectively split by the new method and pre- 
liminary sequence data have been obtained. 

It has been demonstrated that the imidazole ring 
undergoes smooth oxidative cleavage with N- 
bromosuccinimide to a keto-aldehyde, formic acid, 
and two moles of ammonia. In the course of ef- 
forts to protect the imidazole ring from cleavage 
it was found that the ring may be selectively acyl- 
ated with various sulfonyl halides at pH 5.5 to 
give sulfonyl imidazoles which are resistant to 
oxidative cleavage but are reconvertible to imidaz- 
oles under relatively mild hydrolytic conditions. 
This newly discovered phenomenon has advanced 
the chemistry of histidine along several fronts: 
(1) A novel protective group for the synthesis of 
histidyl peptides has become available, (2) the 
imidazole ring may be selectively and reversibly 
blocked in enzymes to determine the contribution 
of histidine to active catalytic sites, (3) the imid- 
azole ring can be protected in the course of oxida- 
tive degradation of tyrosine and tryptophan- 
peptide bonds and can be subsequently recovered. 

In a collaborative study with the NHI an ap- 
proach to a topographic neurochemistry of the 
brain has been started. Only certain regions of 
the brain, such as hypothalamus and caudate nu- 
cleus, but not cerebellum, have been found to con- 
tain the specific enzyme, dopamine-/3-oxidase, that 
converts dopamine to norepinephrine. Boiled 
brain tissue produced only a "norepinephrine- 
like" fraction identified as the "isographic" and 
isomeric 2,4,5-trihydroxyphenethylamine, an iso- 
mer of trisnormezcaline, arising not by enzymatic 
hydroxylation of the side chain, but by non-enzy- 
matic nuclear hydroxylation. The existence of 
this new isomer of norepinephrine, having all of 
its chromatographic properties, has necessitated 
the reevaluation of a large body of previous 
studies on the biosynthesis of norepinephrine in 
other laboratories. 



On administration of radioactive dopamine to 
animals, up to 1 percent of radioactive trihy- 
droxyphenethylamine can be isolated from urine. 
This is a minimum conversion value since only 1 
percent of administered trihydroxyphenethyl- 
amine is recovered from urine. The new metab- 
olite, as preliminary pharmacological studies 
indicate, is more active in certain systems than 
dopamine. Its endogenous formation remains to 
be shown. 

It has now been found that the new enzyme in- 
volved in the inactivation of catecholamines, i.e. 
catechol-O-methyltransferase, converts p-substi- 
tuted catechols not only to m-O-methyl, but also 
£>-0-methyl derivatives. The ratio of m- and p-O- 
methylation is dependent on the electronegativity 
of the para substituent, and the pH at which enzy- 
matic methylation is carried out. For example, 
3,4-dihydroxyacetophenone is methylated enzy- 
matically to the extent of 40-60 percent at the p- 
hydroxyl. Dopamine is converted to 10-15 
percent of the p-O-methyl derivative. The same 
conversions in vivo lead to lower ratios of para- 
methylated products, because enzymatic demethyl- 
ation by a microsomal TPN-requiring system 
proceeds faster with p-O-methyl than with m-O- 
methyl catechol ethers. A novel type of "trans- 
methylation" is the in vivo conversion of a 
2?-0-methyl to a m-O-methylcatechol, namely, the 
interconversions of acetovanillone^±acetoisovanil- 
lone, a type of compound recently isolated from 
adrenal extracts. Epinephrine and norepine- 
phrine gave 10 percent "paranephrine" and "nor- 
paranephrine" in vitro, but not in vivo. 

In collaboration with LPT, the effect of the se- 
lective oxidation of tryptophan on the enzymatic 
activity of trypsinogen, acetyltrypsinogen, an en- 
zymatically active fragment of trypsinogen and 
trypsin has been explored. The marked difference 
in reactivity of tryptophan in trypsin and tryp- 
sinogen is ascribed to differences in their second- 
ary or tertiary structure. Enzymatic inactivation 
(trypsin) or less of activatability (trypsinogen) 
was studied as a function of the oxidative modi- 
fication of tryptophan. Such partially inacti- 
vated enzyme preparations still had their active 
phosphorylation sites intact. At least one trypto- 
phan residue is needed for activity. This demon- 
strates that an intact phosphorylation site per se 
is not sufficient for enzymatic activity but that 



112 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



additional sites, such as an intact indole nucleus, 
are necessary. This points to a possible separa- 
tion of the sites of activity and specificity which 
may be put to further use for the study of enzyme 
mechanisms. 

A rapid synthesis of kynuramine from trypta- 
mine has made possible the observations in the 
NHI that kynuramine is probably not another 
biogenic amine, but that it is an excellent substrate 
for monoamine oxidase. The aldehyde produced 
by this enzyme undergoes mtframolecular cycliza- 
tion to the end product 4-quinolone before any oxi- 
dation to the acid takes place. Thus, kynuramine 
is an ideal substrate for the rapid routine assay of 
monoamine oxidase activity in tissues without get- 
ting interference from DPN. 

A cooperative study with the Laboratory of 
Clinical Biochemistry, NHI, has demonstrated the 
competitive inhibition of O-methyltransferase. 
The potentiation of epinephrine by pyrogallol had 
been interpreted 30 years ago as an antioxidant 
effect. It has now been shown that competitors of 
methyl transferase, such as pyrogallol, catechol, 
glycocyamine, 3,4,5 - trihydroxyphenethylamine 
(trisnormezcaline) inhibit the inactivation of 
norepinephrine by methylation to the extent of 
40-100 percent. By contrast, the half-life time 
of norepinephrine is not affected by typical mono- 
amine oxidase inhibitors such as marsilid. 

The study of the kinetics of the decarboxyla- 
tion of substituted ??-hydroxycinnamic acids has 
adduced evidence for the intermediate existence of 
quinonemethines as the species undergoing decar- 
boxylation, an observation of significance for the 
evaluation of similar metabolic processes. 

New staining reagents for the histochemical de- 
tection of proteolytic enzymes have been devel- 
oped in collaboration with the Laboratory of Path- 
ology. By these techniques, a new trypsin-like 
enzyme has been found to occur almost exclusively 
in mast cells, which differs from trypsin in not 
requiring activation. 

A new tool, nuclear magnetic resonance spec- 
troscopy, has been applied to several problems of 
structure determination during the year. It has 
revealed the position of the double bond in dehy- 
droproline, the position of the hydroxyl in an 
isomer of hydroxyproline, the location of the 
acetyl groups in partially acetylated derivatives 
of deoxystreptamine, the course of bromination of 



certain indoles, the location of the bromine in 
brominated lysergic acid diethylamide, and the 
absence of ring-chain tautomerism in various 
tryptamine derivatives. 

A novel bound form of the neurotropic y-ami- 
nobutyric acid in brain has been discovered in the 
Laboratory of Clinical Biochemistry, NHI. A 
collaborative study led to its identification as 
homocarnosine, i.e., y - aminobutyrylhistidine, 
which was also obtained by synthesis. 

Steroids. Three new crystalline substances have 
been added to the list of compounds isolated from 
fecal lipids. Two of them may be hydroxylated 
fatty acids and the third a new sterol. 

A fascinating collaborative project of the Ster- 
oid Section with an NHI microbiologist culmi- 
nated in the identification of acrasin, a factor or 
hormone involved in the differentiation of slime 
mold, as a derivative of stigmasterol. This suc- 
cess was possible only on the basis of previous im- 
provements in the identification of steroids on a 
micro scale in connection with studies on fecal 
lipids. 

In the reinvestigation of the nonsaponifiable 
fractions of the tapeworm, it was found that in 
Taenia taeniae/ ortnis and in Moniezia sp. choles- 
terol constitutes by far the most prevalent un- 
saponifiable substance (98 and 85 percent). 

In order to study the biogenesis of sapogenins, 
slices of Dioscorea, the Mexican yam, the major 
source of the Western Hemisphere's supply of 
partially synthetic cortical steroids, have been in- 
cubated with radioactive mevalonic acid. The 
latter has been converted into four radioactive 
products whose identification is in progress. 

In work toward the elucidation of the structure 
of pennogenin, it was found that the lithium hy- 
dride reduction of 22,26-oxido-A 17(20) -cholestene- 
3/3,22-diol-16-one yields 22,26-oxido-A 17(20) -choles- 
tene-3/?,16£-diol (I) and not A 17(20) -22-isoallospiro- 
sten-3/3-ol as formerly believed. The A 5 series of 
compounds also gave analogous results. The cata- 
lytic reduction of I leads to the formation of the 
hitherto unknown l7a-cholestane side chain. 
17-Isocholestane would be of value as a reference 
compound. 

Solasodine, the aglycone of a number of widely 
occurring solanum alkaloids, has now been suc- 
cessfully degraded in very good yields (65 per- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



113 



cent) to the acetate of pregnadienolone, the start- 
ing material from which most of the biologically 
active steroids are obtained. The method consists 
in the acid catalyzed rearrangement of 0,N-di- 
acetyl solasodine to the pseudo derivative, fol- 
lowed by oxidation and solvolytic cleavage of the 
acylester side chain. A semicontinuous procedure 
has also been developed. This process has at- 
tracted much attention in the Eastern hemisphere 
where solanum alkaloids (and not sapogenins) 
serve as starting material for partially synthetic 
cortical steroids. 

The stereochemistry of the six- and five-mem- 
bered (C/D) ring juncture in steviol and 
isostevoil, the aglycones from stevioside, one of 
the most potent natural sweetening agents occur- 
ring in a Peruvian shrub, has been established. 
Furthermore, the two epimeric dihydrosteviols 
from stevoil and isosteviol have been converted in 
an eight-step degradative process to (-)a-dihydro- 
kaurene and (-)/J-dihydrokaurene, two naturally 
occurring terpenoid derivatives from New Zea- 
land. With the exception of a few minor points, 
the structure of stevoil and isosteviol has been 
established. 

In continuation of the search for corticoid- and 
sex-hormone antimetabolites and carcinostatics, 
A 4 -androstene-9a-thiocyano-3,ll,l7-trione has been 
converted to the corresponding 9a-thiocarbamide 
and 9a-thiol. Similarly, 9a-thiocyanocortisone 
has been converted to the corresponding 9a-thio- 
carbamide. A new route to ll/?-mercaptocorti- 
coids has been opened through the synthesis of 
3,9a - epoxy - ll/J-thiocyano-5/?-pregnan-3/3,17a,21- 
triol-20-one. 

In connection with studies on the chemical con- 
version of steroidal hormones and vitamins to 
possibily physiologically active derivatives of an- 
thracene, two new isomers of anthroergosterol and 
anthracholesterol have been obtained from the cor- 
responding A 5,7>9(11) -trienic sterols by treatment 
with p-toluene sulfonic acid in chloroform. Their 
chemical structures and relationship to the Nes- 
Mosettig rearrangement are being studied. 

In order to refine the methods for routine anal- 
ysis of clinically important steroids the separation 
and quantitative estimation of a number of 17- 
ketosteroids by gradient elution chromatography 
have been elaborated and further work is continu- 
ing in this direction. 



In cooperation with NCI, rat adrenal tumor is 
being analyzed for adrenal cortical hormones. 

Office of Mathematical Research 

General. The Office of Mathematical Research 
has the broad objective of contributing to theo- 
retical biology as a biological subscience ; produc- 
ing concepts and theoretical apparatus for the ra- 
tional analysis and quantitative interpretation of 
biological problems; and furnishing consultation, 
aid and advice to the subject-matter scientists of 
NIH. Much importance attaches to stimulating 
experimental biology by invoking new points of 
view, producing theories of practical predictive 
value, and formulating deductive models which 
can give purposeful direction to further experi- 
mentation. But it is held to be equally impor- 
tant to contribute to sound "philosophy," to deal 
with abstract problems on their proper mathemat- 
ical ground and to operate at a level of general- 
ity not necessarily dictated by immediate and ob- 
vious applications in biology, medicine, and pub- 
lic health. 

Research. Work has continued on the develop- 
ment of mathematical and computational meth- 
odology for the analysis of tracer data. Pro- 
grams have been written for parameter estimation 
in compartmental systems. These programs, 
available to any investigator, are sufficiently gen- 
eral to cover a broad spectrum of models and to 
accept, as input, experimental data in any one of 
a variety of forms. Methods have been developed 
for obtaining the variances and covariances of 
such estimates so that the significance of observed 
changes, introduced for example by a given treat- 
ment, can be assessed. These programs can be 
applied to any problem in the realm of "linear 
kinetics." In particular, they have been applied 
to C-14-labeled glucose data on human subjects 
and in a comprehensive continuing study of 
patients having various thyroid abnormalities and 
subjected to various treatments. In both in- 
stances suggested additional experimentation has 
come out of the mathematical analysis. Work 
has been underway and will continue on the prob- 
lem of taking into quantitative account, in the fit- 
ting of data on a given compartment, information 
which is available on one or more other compart- 



114 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



merits, studies of consistency checks, and the ex- 
ploitation of redundant data in the uniqueness 
question. 

The problem of the spread of electric current 
from a neuron soma into branching dendritic trees 
has been formulated and solved. Analysis of cur- 
rent experimental data indicates that motoneuron 
dendrites play a dominant role in determining 
motoneuron properties and this is in direct con- 
trast to what had been assumed in the absence 
of an appropriate mathematical solution. Signifi- 
cant progress has been made on the general prob- 
lem of membrane-potential spread over soma and 
dendrites in response to synaptic current genera- 
tion with an arbitrary time course and spatial 
distribution. The theory permits distinction be- 
tween synaptic potentials generated predominant- 
ly in the dendrites and predominantly on the 
soma. Recent experimental techniques and sev- 
eral ambiguities in the current theories of synaptic 
excitation are being analyzed within the frame- 
work of these theoretical results. The above gen- 
eral analysis has also led to practical estimation 
procedures for motoneuron time constants and 
soma-dendrite conductance ratios. A procedure 
has been devised and programmed for the compu- 
tation of external potential fields around a neuron. 
Application of this program will enable the 
mapping of isopotential contours and quantitative 
assessment of certain approximations which have 
been standard in neurophysiology. 

Work has continued on the problem of specify- 
ing the area under the curve in terms of the co- 
efficients of the differential equation of which that 
curve is a solution. Special cases of the general 
theorem have found extended application in the 
analysis of the inhibition of thrombin formation 
by soybean trypsin inhibitor, in the estimation of 
rate constants in systems for which no analytical 
solution of the rate equation is known, and is the 
choice of kinetic models based solely on final 
yields. This same problem has appeared in the 
stochastic theory of absolute reaction rates and it 
has been shown that the results employed there, 
subject to severe restrictions in fact, apply to a 
very general class of matrix. Work has continued 
on matrix theorems as applied to linear analysis 
in general, relaxation time analysis of systems 
close to equilibrium, and the relation between ir- 
reversible thermodynamics and chemical kinetics. 



Other Activities. This office has continued to 
furnish extensive consultation and collaboration 
to other scientists at NIH. During the past year, 
such activities have involved members of the Re- 
search Associate Program. A member of this 
office is giving the mathematics course in that 
program and at their request is supervising an 
applied mathematics seminar for the Research 
Associates. Members of this office have served 
on committees (as well as in less formal advisory 
roles) for NIAMD, NINDB, NIMH, Office of Re- 
search Planning, DRG, DGMS, and Heart Con- 
trol Program, PHS. Members of this office pre- 
sented an entire symposium on Mathematical 
Models in Biology (jointly sponsered by the Bio- 
metrics Society and AAAS). Further external 
interests in the activities of this office are indi- 
cated by the following invited one hour lectures : 
Two at an IBM Symposium on Computers in 
Medicine, one for the American Mathematical 
Association, and one before a joint symposium 
sponsored by the American Statistical Associa- 
tion and the Biometrics Society. 

Concluding Comments 

The vigor of a research organization is in good 
part determined by the flux of scientists in and 
out. Its status in the community of such institu- 
tions is likewise measured by this flux. Through- 
out the year, a sizable number of visiting scien- 
tists have been in attendance in the laboratories 
and clinical facilities of NIAMD, these ranging 
from senior and world-renownel investigators to 
select and exceptional younger scientists, hope- 
fully leaders of the future. Among the more dis- 
tinguished of these visitors have been Drs. 
Gustafsson of Lund, Hestrin of Jerusalem, Pitt- 
Rivers of London, Tjio of Saragossa, Watson of 
Minneapolis. Their terms of service at Bethesda 
have ranged from 1 to 12 months, and both the 
visitors and the permanent staff have profited 
enormously from these arrangements. In addi- 
tion a considerable number of very able guest 
workers have elected to spend part or all of their 
available time at NIAMD. The avidity with 
which such guests and visitors seek out this in- 
stitute is taken as an indication of the excellence 
of our staff and our facilities. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



115 



By way of compensation to the scientific com- 
munity, a number of NIAM D scientists have been 
provided with the opportunity to work and spend 
time in other laboratories in America and abroad. 
The 11 members of our staff who have spent sig- 
nificant periods of time in other laboratories 
during the past calendar year have worked at 
such centers of scientific activity as Woods Hole, 
Paris, Copenhagen, Mill Hill (London), and 
Osaka. We believe this exchange to be extremely 
fruitful, bringing to Bethesda foreign ideas and 
techniques. Also it serves to familiarize our 
foreign colleagues with recent scientific progress 
in this country. We further believe that this flux 
of persons to and from Bethesda is almost as essen- 
tial to the continued vigorous pursuit of our 
mission as is the flux of fluids across cell mem- 
branes to the continued survival of the organisms 
we study. 

Another essential feature of organic survival is 
growth, and, in the necessarily intellectual at- 
mosphere of NIAMD, growth connotes learning 
and teaching. Over and above the process of 
mutual education which is inevitable when many 
scientists work together, a consciously designed 
teaching program is now well under way. The 
first class of research associates graduated in June 
1959; the third class was admitted at that time. 
This comprizes four new members, young phy- 
sicians desirous of getting concentrated training 
in research. Many more applications are filed by 
candidates than can be accommodated, and the 
quality continues to be the highest. The program 
is popular both with the associates and with their 
preceptors. Additional activities have been the 
provision for seven fellows in the CO STEP pro- 
gram and an increasing interest in the laboratory 
and academic graduate education of secondary- 
school science teachers. In the latter capacity, we 
have housed, for brief periods, four high-school 
teachers and four students. NIAMD scientists 
collaborated with the staff of NCI in an intensive 
1-day program designed to give high-school 
teachers an appreciation of contemporary bio- 
medical research as practiced at Bethesda. 

From these activities it will be seen that, in ad- 
dition to fulfilling its committed obligation of pro- 
ducing publishable research, NIAMD has, in 
limited degree, assumed the responsibility of pro- 
ducing men. It may be that in the long light of 



history, the men which it produces will be as im- 
portant a contribution to the community at large 
as is the scientific research which its scientists 
conduct. 



CLimCAL INVESTIGATIONS* 

The Clinical Investigations Unit has grown 
conservatively and according to plan in several 
functional areas : clinical research program; num- 
ber of patients and varieties of diseases studied; 
scientific publications ; and number of staff scien- 
tists, visiting scientists and guest workers. This 
growth has been encompassed without any struc- 
tural (laboratory space) expansion. 

One new branch, Pediatric Metabolism, and two 
new groups, gastroenterology and epidemiology of 
arthritis and rheumatic diseases have been added. 
Pediatric Metabolism Branch, under the super- 
vision of Dr. Paul A. di Sant'Agnese, will study 
three childhood diseases : fibrocystic disease of the 
pancreas, glycogen storage diseases and celiac 
disease. The programs of the other new groups 
are described below. 

A total of 389 inpatients was admitted during 
the 12-month period from December 1, 1958, to 
November 30, 1959, an increase of 55 over a simi- 
lar period last year. The total patient days were 
16,942, an increase of 690 over the preceding year. 
In the admissions and followup, 1,520 patients 
were examined and studied, an increase of 325 
over the past year. The average inpatient stay 
at the Clinical Center was 43 days. The bed ca- 
pacity increased from 59 to 65 during the current 
year. An average census of 79 percent was main- 
tained. As in the preceding three years, the in- 
patients on the service of the National Institute of 
Dental Research were accommodated on NIAMD 
units. 

Clinical and laboratory investigations related to 
the diseases studied at NIAMD have resulted in 
72 publications in scientific journals, monographs, 
annual reviews, and medical textbooks. During 
the past year, Dr. J. Edward Rail was given the 
Arthur H. Flemming Award, Dr. Joseph J. Bunim 



* Prepared by Joseph J. Bunim, M.D., Clinical Director, 
NIAMD. 



556044— 6C 



116 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



was President of the American Rheumatism Asso- 
ciation and Cochairman of the Pan American 
Congress on Rheumatic Diseases. Dr. Bunim was 
appointed Clinical Professor of Medicine at 
Georgetown University. Dr. Paul A. di Sant'Ag- 
nose was given an award by the National Cystic 
Fibrosis Foundation. 

At the request of the Bureau of Medical Services 
of the TJSPHS, members of the Clinical Investi- 
gations staff gave lectures and conducted ward 
rounds at the following Public Health Service 
Hospitals : Baltimore, Seattle, Staten Island, and 
Savannah. 

Clinical investigation on selected ambulatory 
patients was conducted by staff members at three 
"outside" specialty clinics in regional hospitals: 
Dr. Roger L. Black at the Arthritis Clinic of D.C. 
General Hospital, Dr. Kurt J. Bloch at the Arth- 
ritis Clinic of Georgetown University Hospital, 
and Dr. James B. Field at the Diabetes Clinic of 
Georgetown University Flospital. 

Our staff scientists have derived considerable 
benefit from the association of distinguished visit- 
ing scientists and guest workers, who have come to 
work here during the past year : Dr. Cecil J. Wat- 
son, Professor of Medicine, University of Minne- 
sota; Dr. Rosalind Pitt-Rivers from the Medical 
Research Council, Mill Hill, London; Dr. Anne- 
Marie Hof er from Frankfurt, Germany ; and Dr. 
Samuel Rose, Professor of Physiology, University 
of Melbourne, Australia ; Dr. Panu Vilkki, a visit- 
ing Fellow from Turku, Finland. The guest 
workers were: Dr. John Worthington, Jr., sent 
here by the Mayo Clinic; Dr. Kingsley Mann, 
sent here by the Upjohn Company; and Dr. 
Evelyn Hess, Fellow, Empire Rheumatism Coun- 
cil of England. 

Arthritis and Rheumatism Branch 

Research in arthritis and connective-tissue 
diseases has taken new direction as a result of 
recent studies done at NIAMD and other institu- 
tions, and of new immunological and genetic 
concepts advanced in this country (Lederberg, 
Billingham), Australia (Burnet), England 
(Medawar), and Sweden (Grubb). Some of the 
recent observations and provocative ideas that 
have opened new trails of investigation can be 
mentioned only for orientation in this brief report. 
The rheumatoid factor, a macroglobulin (19S), 



has been isolated in other laboratories and at 
NIAMD, and its chemical composition and physi- 
cal properties denned. We are in a better position 
now to wrestle with the important questions: Is 
the rheumatoid factor a true antibody and, if so, 
what is the specific antigen? Is the rheumatoid 
factor, whatever its biologic nature may be, a by- 
product or does it have pathogenetic implications? 
Evidence has been collected which strongly sug- 
gests a close relationship between rheumatoid 
arthritis and systemic lupus erythematosus 
(S.L.E.). The L.E. factor, consisting of light 
(7S) gamma globulins, is composed of multiple 
antibodies to several components of cell nuclei. 
This suggests that S.L.E. may be another example 
of an autoimmune disease in man. The L.E. fac- 
tor is found more frequently in serum of patients 
with rheumatoid arthritis than in any other dis- 
ease except S.L.E. itself. Patients with congeni- 
tal and acquired agammaglobulinemia develop 
rheumatoid arthritis far more frequently (25%) 
than does any other human population. Since 
gamma globulin is deficient in these patients, both 
the rheumatoid factor and the L.E. factor are, of 
course, absent. But many relatives of these pa- 
tients have either the rheumatoid factor or L.E. 
factor or, less frequently, rheumatoid arthritis or 
systemic lupus erythematosus. Sir Macfarlane 
Burnet and Joshua Lederberg have separately 
postulated that "each immunologically competent 
cell, as it begins to mature, spontaneously produced 
small amounts of antibody corresponding to its 
genotype; that the stereospecific segment of each 
antibody globulin is determined by a unique se- 
quence of amino acids ; and that the cell making a 
given antibody has a correspondingly unique se- 
quence of nucleotides in a segment of its chromo- 
somal DNA — its 'gene for globulin synthesis.' 
The mature cells proliferate extensively under 
antigenic stimulation but are genetically stable and 
therefore generate large clones genotypically pre- 
adapted to produce the homologous antibody. 
These clones tend to persist after the disappear- 
ance of the antigen, retaining their capacity to 
react promptly to its later reintroduction." 

Clinical research in the Arthritis & Rheumatism 
Branch will include attempts to determine im- 
munological reactivity in patients with rheuma- 
toid arthritis and connective-tissue diseases and by 
epidemiological studies to ascertain whether ab- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



117 



normal serum proteins in patients and their kin- 
ships are genetically controlled. 

STUDIES OF SJOGREN'S SYNDROME 

This syndrome consists of a triad of keratocon- 
junctivitis sicca (KCS), xerostomia and rheuma- 
toid arthritis (r.a.). However, any two of the 
three features justify the diagnosis of Sjogren's 
syndrome. Twenty-one patients with this rela- 
tively rare syndrome have been intensively stud- 
ied during the past year. The diagnostic criteria 
of KCS and xerostomia have been established and 
verified in all our patients by staff members of 
the Ophthalmology Branch of NINDB and NIDR 
respectively who actively collaborated in this 
project. About one-third of the patients had 
Sjogren's syndrome without arthritis, a few pa- 
tients had systemic scleroderma (a connective-tis- 
sue disease) but not rheumatoid arthritis, and the 
rest had Sjogren's syndrome with classical r.a. 
Yet every patient but one (even those without 
r.a.) had the rheumatoid factor and two had the 
L.E. factor. All 21 patients had hyperglobu- 
linemia, 75 percent hypergammaglobulinemia, 60 
percent had antinuclear antibody demonstrated by 
immunofluorescence, and 25 percent had positive 
direct Coombs' test. Biopsies of the salivary 
glands done in 10 patients showed in each case typ- 
ical histopathological changes consistent with 
Sjogren's syndrome. These lesions bear a strik- 
ing resemblance to the histological alterations of 
the thyroid gland in Hashimoto's disease which 
is believed to be an autoimmune disorder. 

Our studies reveal that Sjogren's syndrome is 
a variant of r.a. in which an exalted immune re- 
activity is exhibited. Prospective investigation 
will consist of two types of study : (1) An attempt 
to find an antibody to the patient's tissue compo- 
nents and (2) using the 21 patients as propositi, 
kinships will be studied by the epidemiology 
group to determine the pattern of abnormal serum 
proteins and the occurrence of rheumatic diseases 
in these families. 



EPROEMIOLOGY OF RHEUMATOID- AND 
OSTEOARTHRITIS 

This study was initiated intramurally by the 
Arthritis and Rheumatism Branch of NIAMD 



during the past year. The prevalence of both 
types of arthritis was studied in the following 
population groups : Alaskan Eskimos (457), Alas- 
kan Indians (182), U.S.A. whites (436), and Eng- 
lish whites in Wensleydale (489). The diagnosis 
was based on three kinds of evidence: (1) clinical 
findings (history and physical examination), (2) 
X-rays of hands and feet, and (3) serological ex- 
amination for rheumatoid factor by the bentonite 
flocculation test and the sheep-cell agglutination 
test. 

The prevalence of osteoarthritis in Eskimo 
males and females in each decade was less than in 
U.S.A. whites. The difference was statistically 
significant in the North and South Eskimo males 
above age 40 (p = .001) and in the North Eskimo 
females of the same age group. The difference 
was not significant in the South Eskimo females. 
Analysis of the data on the other population 
groups has not yet been completed. 

MECHANISM OF ACTION OF STEROIDS AT 
MOLECULAR LEVEL (IN VITRO) 

DPNH Oxidation. Low concentrations of vari- 
ous hormonally active steroids and stilbesterol in- 
hibit DPNH oxidation (but not TPNH oxida- 
tion) by enzyme preparations from a number of 
sources (noncompetitive with DPNH). The ef- 
fect is catalytic and not related to steroid metabo- 
lism. In addition to variations in sensitivity from 
organ to organ, there is a 1,000-fold variation in 
range of potency among the active steroids. In 
kidney, the Ki (or half -maximum inhibitory con- 
centration) for stilbesterol is 8X10 -7 M. This 
means that a measurable effect can be observed 
with concentrations as low as .02 gamma/cc. 
Cholesterol and tetrahydro E are ineffective. The 
effect has been studied in brain, spleen, muscle, 
heart, liver, thymus, and kidney of the rat. Beef 
pituitary is also under study. 

Extension of studies to include preparations 
from E. coli, B. subtilis, and yeast (S. fragilis) 
revealed a similar inhibition of DPNH oxidation, 
and this could be correlated with known effects of 
steroids on growth. The effect was also demon- 
strated in preparations from Ehrlich and S-37 
mouse ascites tumor. 



118 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Further refinement of studies has revealed the 
site of inhibition to be DPNH-cytochrome C re- 
ductase (specifically between flavoprotein and cy- 
tochrome b), a major link in the chain of hydro- 
gen transfer in the cell's oxidative reactions. In- 
terestingly, the inhibition is competitively re- 
versed by tocopherol. This suggested a need for 
investigation into a possible relationship between 
steroid and vitamin E. Examination of the tis- 
sues of vitamin E-deficient rats, however, did not 
reveal differences in DPNH cytochrome C re- 
ductase or the degree of steroid inhibition. 

EFFECT OF CERTAIN COMPOUNDS ON 
URIC-ACID SYNTHESIS IN MAN 

The metabolic origin and disposition of uric 
acid in gouty patients has been studied further 
by administering isotopically labeled precursors 
of uric acid along with labeled uric acid itself and 
following the incorporation of label into urinary 
uric acid in normal and gouty subjects. A sub- 
stantial portion of the patients with gout show an 
excessive synthesis de novo of uric acid as meas- 
ured by the extent of incorporation of glycine- 1- 
C 14 into urinary uric acid. Additional patients 
can be shown to be producing excessive amounts 
of uric acid if the glycine incorporation data is 
corrected for the dynamics of the urate pool. 
This increased production does not show up in 
the urinary uric-acid excretion values because of 
its extrarenal disposition. There still remains a 
portion of the gouty patients who show no demon- 
strable difference in the extent of glycine-1-C 14 
incorporation into urinary uric acid from that of 
normal individuals. 

A pharmacological agent which suppresses the 
excessive uric-acid synthesis found in some gouty 
subjects has been studied further. 6-Diazo-5-oxo- 
L-norleucine (DON) which has been shown in 
this laboratory to suppress uric-acid synthesis in 
two gouty subjects has been administered to a 
total of seven gouty patients. Two patients who 
showed no drop in serum uric acid or in urinary 
uric-acid excretion nevertheless showed a sub- 
stantial reduction in the incorporation of glycine- 
1-C 14 into urinary uric acid. This suppression of 
purine biosynthesis was evidently masked by the 
large urate pool in these subjects. Undesirable 
effects of DON consisted of duodenal ulcers in 



two patients and ulcerations of the oral mucosa in 
five of the seven patients studied. Routine use 
of this drug for suppressing the uric acid produc- 
tion in gouty patients appears to be imprudent. 
It is conceivable, however, that more specific in- 
hibitors of purine biosynthesis might carry with 
them a more favorable therapeutic index. 

An experimental tool for studying the home- 
ostatic control of purine synthesis in the human 
has been found in the action of a drug, 2-ethyl- 
amino-l,3,4-thiadiazole, a nicotinamide antagonist 
which has been used experimentally in the treat- 
ment of cancer. Other workers noted that its use 
in the human resulted in an increase in both serum 
urate values and in daily urinary uric acid. The 
origin of this increased uric acid, whether from 
cellular breakdown or de novo synthesis, was not 
clear. We have been able to confirm this finding 
and furthermore to show that the increased uric 
acid production is the consequence of an increased 
purine biosynthesis induced by 2-ethylaminothia- 
diazole. The extent of incorporation of glycine- 
1-C 14 into urinary uric acid in a non-gouty in- 
dividual was brought up to the range observed in 
gouty subjects by administration of 2-ethylamino- 
thiadiazole. Furthermore, its effect was complete- 
ly prevented by administration of large doses of 
nicotinamide. This drug was found to have a 
comparable effect on urinary allantoin and uric- 
acid production in the guinea pig and in vitro 
studies of its action are now underway. 

NEW URICOSURIC AGENTS IN GOUT 

Studies on drugs for the management of prob- 
lem cases of gout have been continued. Previous 
work in the National Heart Institute on a group 
of compounds chemically related to phenylbu- 
tazone had shown that antirheumatic activity 
could be correlated with chemical structure and 
that uricosuric activity could be related to the 
acid association constant (pKa) of the drug. A 
urinary metabolite of phenylbutazone, oxyphen- 
butazone, had been shown to possess potent anti- 
rheumatic activity, but very little uricosuric ac- 
tivity. Upon the introduction of a keto group 
into the side chain of oxyphenbutazone, the re- 
sulting compound (G-29701) was found to possess 
potent uricosuric activity, but little antirheumatic 
activity. Again the uricosuric activity was cor- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



119 



related with an increased acidity of the compound 
with the pKa dropping from 4.6 for oxyphen- 
butazone to 2.3 for keto-oxyphenbutazone. There 
was a corresponding decline in the biological half- 
life from 72 hours to 8 hours, which prevented 
the high-serum levels needed for an antirheumatic 
effect with the parent compound. This drug has 
been given to nine patients for short periods of 
time and has been very well tolerated, with no 
toxic side effects to date. Since the other potent 
uricosuric agents now available have a considera- 
bly shorter biological half -life, on the order of 3 
hours, the 8-hour half-life of keto-oxyphenbu- 
tazone gives promise of providing a more sus- 
tained uricosuric action with less frequent admin- 
istration of the drug. 

The antagonistic action of salicylates on the 
uricosuric effect of zoxazolamine and sulfinpyra- 
zone has been further studied and found to exist 
at even low doses of salicylates. By contrast, 
acetaminophen, which is the active metabolic 
product of acetanilid and acetophenetidin, gives 
no such antagonistic action, while at the same 
time providing an analgesic action. 

ENZYMIC AND CLINICAL STUDIES ON IN- 
BORN ERRORS OF METABOLISM OTHER 
THAN GOUT 

Alcaptonuria and Ochronotic Arthritis 

Objectives in studying patients with alcaptonu- 
ria have been several: (1) to determine the exact 
nature of the metabolic defect in this condition, 
(2) to study the hereditary pattern of this dis- 
ease, and, if possible, to develop a test which will 
detect the heterozygous state in relatives of alcap- 
tonurics carrying the trait, (3) to study the for- 
mation and deposition of the pigment derived 
from homogentisic acid and to determine how it 
produces the pathological changes in the connec- 
tive tissues, particularly the joints, (4) to study 
the cause of the arthritis nearly always associated 
with this condition, and (5) to attempt various 
means of treatment of this metabolic disease. 

Nature of Defect in Alcaptonuria. Quantita- 
tive analysis of the enzymes involved in tyrosine 
metabolism has been made in liver and kidney 
homogenates from autopsy specimens of another 
patient with alcaptonuria. Again it has been pos- 



sible to show that alcaptonuric tissues differ from 
the normal only in having no detectable homo- 
gentisic-acid oxidase activity. Thus, it has been 
clearly demonstrated in two families that the de- 
fect in this metabolic disease consists of a defi- 
ciency of homogentisic-acid-oxidase activity in 
the liver and kidney. 

Inheritance of Alcaptonuria. The recent sug- 
gestions of Milch and Milch that this disease is 
inherited as a dominant trait with incomplete pen- 
etrance, rather than as a simple autosomal reces- 
sive inheritance, is not supported by an examina- 
tion of the pedigree of one of our patients. Upon 
careful questioning it was disclosed that there had 
been a consanguineous marriage which was not 
mentioned in earlier interviews. With this com- 
plete information, a simple recessive inheritance 
adequately explains the expression of the disease 
in this family. 

Experimental Ochronosis and Arthritis in 
Guinea Pigs. As part of the study on the mech- 
anism by which the accumulation of homogentisic 
acid leads to the development of ochronotic ar- 
thritis in alcaptonuria, the distribution of homo- 
gentisic acid in the tissues of guinea pigs has been 
measured at different times by a specific enzymatic 
method after the intraperitoneal injection of this 
acid. Very low concentrations were found in the 
muscle, liver, and the other organs, but values 
almost as high as in plasma were present in the 
cartilage and skin. This unusual predilection of 
homogentisic acid for the connective tissues is in 
agreement with the deposition of the ochronotic 
pigment in the same areas. Further studies are 
being made of the nature and syntheses of the 
pigment and its relationship to the associated 
arthritis. 

Enzymatic Synthesis of Homogentisic Aero 
(Role of Vitamin C in Tyrosine Metabolism). 
Detailed studies of the enzyme system of liver 
which catalyzes the formation of homogentisic 
acid from p-hydroxyphenylpyruvic acid (the keto 
acid of tyrosine) are being continued. Vitamin 
C is involved in this enzymatic reaction and scor- 
butic guinea pigs have a defect in tyrosine me- 
tabolism and excrete p-hydroxyphenylpyruvic 
acid when fed this amino acid. The metabolic 
defect is corrected by vitamin C, but until recently 



120 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



it was not known how this vitamin maintains nor- 
mal tyrosine metabolism. Some insight into the 
mechanism came in studies with purified liver 
enzymes. It was found that ascorbic acid and 
2,6-dichlorophenolindophenol had the property of 
protecting one of them, p -hydroxy phenylpyruvic 
acid oxidase (the enzyme which catalyzes the oxi- 
dation of p-hydroxyphenylpyruvic acid to homo- 
gentisic acid) from being inhibited by its sub- 
strate. In the presence of ascorbic acid, oxidation 
continued; in its absence, the oxidation slowed 
down and stopped. Recently we have been able 
to demonstrate the way the vitamin acts in vivo. 
Scorbutic guinea pigs were found to have as much 
p-hydroxyphenylpyruvic acid oxidase as normal 
animals, but when the scorbutic group was in- 
jected with p-hydroxyphenylpyruvic acid, over 
half of their liver p-hydroxyphenylpyruvic acid 
oxidase was inactive one hour later. In contrast, 
injection of the substrate did not inhibit the oxi- 
dase in normal guinea pigs. It appears that 
ascorbic acid acts in vivo to protect the oxidase 
from inhibition as was found in the enzyme studies 
in vitro. 

Method for the Estimation of Homogentisic 
Aero in Synovial Fluid and Other Tissues. 
The specific enzymatic method to measure small 
amounts of homogentisic acid in plasma has been 
modified to make it suitable for the analysis of 
homogentisic acid in tissues. The method has been 
utilized in studies of the distribution of homo- 
gentisic acid in the tissues of patients with al- 
captonuria and in studies of the distribution of 
this acid in guinea pigs. 

Phenylketonuria 

Early Diagnosis of Phenylketonuria in New- 
born Infant. A sibling born in a family with 
known phenylketonuria has a one in four chance of 
being affected. The new enzymatic method for 
blood phenylalanine developed in this laboratory, 
particularly the micro modification, makes it rea- 
sonable to make the diagnosis within a day or two 
after birth, and such analysis should be done in 
newborn infants in families with known phenyl- 
ketonuria. We are presently doing serial analyses 
on blood samples on an infant with this back- 
ground in order to make the diagnosis and start 
the special diet as soon as possible if this child 
should have phenylketonuria. 



Effectiveness of Diet Low in Phenylalanine 
in Preventing Mental Retardation in Phenyl- 
ketonuric Children. Studies of the effectiveness 
of the low phenylalanine diet in treating phenyl- 
ketonuric children require repeated measurement 
of the level of blood phenylalanine and tyrosine. 
The variable response to the diet, particularly in 
older infants, may be largely due to the greater 
difficulty in maintaining a low level of blood phen- 
ylalanine in this group. We have been analyzing 
the blood each month of several children being 
followed at the Retarded Children's Clinic, 
Georgetown University Hospital, to evaluate the 
effectiveness of the diet. 

GENETIC POLYMORPHISMS IN MAN AND 
OTHER ANIMALS 

Many biochemical traits in humans and other 
animals are genetically determined. Some of these 
may be classified as genetic polymorphisms ; that 
is, the existence in a population of two or more 
easily distinguished forms of a trait, the lesser of 
which could not be maintained by recurrent muta- 
tions alone. The normal hemoglobin-sickle-cell 
hemoglobin system, most of the blood groups, and 
other systems to be described below, fall within 
this classification. From studies in lower animals 
and theoretical considerations, there is reason to 
believe that selection may operate to maintain 
these traits in the incidences found and in some 
cases the selective forces may be related to disease. 
It is of interest to determine if populations living 
under different environmental conditions and 
prone to different diseases have different in- 
cidences of these genes. In addition, studies of 
the distribution of these traits can sometimes pro- 
vide information on genetic relations between sep- 
arated population groups. A field trip to the 
Central Pacific was made in the winter of early 
1959. 

Haptoglobins 

These are a family of serum proteins which bind 
hemoglobin. There are three major patterns in 
humans and these are under genetic control. 
Some rare types have been discovered by an 
NIAMD scientist and other workers. Bloods 
were collected from Micronesians living in the 
Marshall Islands. The prevalence of type 1-1 
was found to be high in the Rongelap people, 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



121 



although not quite so elevated in the smaller num- 
ber of sera studied from the other atolls. Several 
individuals with no haptoglobin were found ; this 
would presumably be of physiological importance 
under some conditions of red-blood-cell break- 
down. In two cases it was found that individuals 
who had no haptoglobin in 1959 had small amounts 
of haptoglobin in 1957. This implies a phenotypic 
change during the course of the two years. 

Monkeys, chimpanzees, and baboons were found 
to have only one of the haptoglobin types and, 
presumably, one of the genes. This implies that 
the polymorphism originated in human popula- 
tions and has been perpetuated by forces of selec- 
tion present in humans but absent in lower pri- 
mates. The haptoglobins have also been studied 
in a variety of animals and interesting species 
differences have been observed. 

Gamma Globulin groups 

The agglutination test for "rheumatoid factor" 
has been used as a diagnostic method for rheuma- 
toid arthritis. It has recently been shown by 
Grubb that the sera of some humans will inhibit 
this reaction, and that the inhibiting material 
travels in the gamma globulin fraction. This in- 
hibiting property is determined by an allelic pair 
of autosomal genes, Gm a and Gm b . A third gene, 
Gm c , which may or may not be at the same locus, 
has also been detected by Steinberg. In a pre- 
liminary study of African, Eskimo, Alaskan In- 
dian, and Micronesian populations, it has been 
shown that the Gm b gene appears to be absent 
from these populations. Studies on the Gm c gene 
are in progress. Furthermore, it was found that 
there is a striking variation in the gamma-globu- 
lin levels in these populations. Thus, some ap- 
parently normal Africans have total gamma glob- 
ulins three times that of normal white Americans. 
The significance of this finding in relation to im- 
munity may be of importance. 

Urinary (3-aminoisobutyric acid (BAIB) 
excretion 

It has been shown by Gartler and others that 
the excretion of BAIB is in part under genetic 
control. Some persons with leukemia and other 
cancers are also high excretors, but the genetic role 
in these cases is not clear. Individuals who ex- 
crete large amounts of BAIB are rare in Euro- 
pean populations. Approximately 200 urine 



samples from Micronesians were studied. It was 
found that nearly 90 percent of these were high 
excretors as compared to 10 percent high ex- 
cretors in white American populations. Some of 
the Micronesians studied had been subjected to 
fallout in 1954 following the detonation of a nu- 
clear device on nearby Bikini atoll. It has been 
shown that radiation can increase the urinary 
BAIB output. However, it is unlikely that this 
is the explanation of the present findings; there 
was no difference between the exposed and unex- 
posed groups, and there was a high prevalence of 
high excretors in a small Micronesian population, 
from a nearby atoll with nearly normal levels of 
radiation. An alternate explanation is that a 
focus of the high excretor genes is present in 
Oceania or Southeast Asia. Studies to determine 
if this is so are in progress. 

Thyroxine-binding proteins of serum 

The serum proteins which bind thyroxine have 
been studied. Four separate binding bands have 
been detected, representing a much more compli- 
cated pattern than had been suspected. These 
bands have been correlated with those seen on 
paper electrophoresis, by the use of two-dimen- 
sional paper-paper and paper-gel electrophoresis 
studies. Variations in the patterns in various 
disease conditions have been studied, and some 
significant alterations noted. Species differences 
in binding patterns have also been found. 

A variation in the position of the fastest mov- 
ing band (the thyroxine-binding pre-albumin) has 
been found in Macaco, mulatta, and this may repre- 
sent a polymorphism. In order to determine if 
this is so, studies on monkey families are contem- 
plated. The relation of these variations in pro- 
tein binding to differences in thyroid physiology 
could be the subject of further study. 

BIOCHEMICAL STUDIES IN DISEASES OF 
THE GASTROINTESTINAL TRACT 

Recent advances in the understanding of disease 
processes in terms of biochemical abnormalities at 
an enzymatic level have made the time opportune 
to explore intensively the gastrointestinal tract 
from this point of view. It is the aim of the newly 
established Gastroenterology Unit to pursue such 
studies at the level of laboratory investigations of 
metabolic pathways in tissue extracts as well as 
at the clinical level. 



122 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The metabolic processes that transpire in the 
cells of the mucosal lining of the stomach, intes- 
tines, and gall bladder are far from completely 
delineated, and consequently their interrelations 
with the physiologic activities of these areas are 
not fully understood. Although the biochemical 
reactions that occur within the liver and pancreas 
have been studied in greater detail, there remain 
many gaps in our knowledge of these organs, too. 
It is the long-term purpose of this unit to investi- 
gate pathways of metabolism of these tissues, us- 
ing at first material from animal sources and even- 
tually biopsy specimens from human subjects with 
and without diseases of these tissues. It is then 
intended to apply the information gained in these 
studies to the further investigation of the func- 
tions of these organs, in animals and humans, and 
to investigations of hormonal regulation of their 
functions. 

Initial efforts are directed toward disease of the 
small intestine. Within this organ food is di- 
gested, and the digestion products are then trans- 
ferred across its wall. The diseases of man in 
which these processes are impaired are known col- 
lectively as the "malabsorption syndome." Pa- 
tients with this syndrome are being studied, as is 
also the process of absorption m vitro, with the 
aim of correlating the information obtained by 
these two approaches in order to understand more 
fully the mechanisms underlying the human dis- 
eases. 

Also being studied are families is which gastro- 
intestinal diseases occur as genetically determined 
traits, in an attempt to discover previously unde- 
tected biochemical aberrations as causal factors 
in the diseases. 

Pediatric Metabolism Branch 

The Pediatric Metabolism Branch will devote 
most of its efforts to the study of cystic fibrosis of 
the pancreas. Other disorders leading to in- 
testinal malabsorption in children and diseases 
due to glycogen storage will also be the object of 
investigation. 

PROPOSED INVESTIGATIONS IN CYSTIC 
FIBROSIS OF THE PANCREAS 

Cystic fibrosis of the pancreas, despite its name, 
is a generalized, hereditary disease of children 



and adolescents in which there is a dysfunction of 
exocrine glands. The fully manifested patients 
have chronic pulmonary disease, pancreatic de- 
ficiency, abnormally high sweat electrolytes, and 
at times cirrhosis of the liver. 

Cystic fibrosis was recognized as a separate 
disease only 20 years ago. At that time most 
patients died in infancy of bronchopneumonia. 
With the advent of effective antibiotic agents and 
with increasing awareness of the disease, hundreds 
of patients are being seen in leading medical 
centers in this country and abroad. The sig- 
nificance of the planned researches becomes ap- 
parent when one realizes that this generalized 
disease is responsible in the pediatric age group 
for the great majority of patients with chronic 
lung disease, for virtually all cases of pancreatic 
deficiency, and for about a third of children with 
cirrhosis of the liver and portal hypertension. 
Cystic fibrosis is now recognized as one of the 
most common chronic diseases of children and a 
leading cause of death in this age group. It is 
estimated that about 25 percent of relatives of 
known patients have some manifestation of the 
disorder and that 5 to 15 percent of the popula- 
tion are carriers of the recessive gene. There is 
reason to believe that cystic fibrosis bears a rela- 
tion to at least some cases of chronic lung disease 
in adults. 

Recent studies, many of them carried out under 
the direction or collaboration of the Branch Chief, 
have shown that there are three defects in exocrine 
secretion in cystic fibrosis that need further ex- 
planation: (1) An abnormality of mucus produc- 
tion, affording a reasonable explanation for the 
pulmonary, pancreatic and hepatic symptoms of 
the disorder. (2) An abnormally high concen- 
tration of electrolytes in eccrine sweat, mixed 
saliva, and tears, a finding which has had great 
importance in changing our concept of the disease. 
The "sweat test" (analysis of perspiration for 
sodium and chloride levels) has become the gen- 
erally accepted method of diagnosis for cystic 
fibrosis, because of its simplicity and reliability. 
It also affords a useful tool for further studies of 
the pathogenesis and genetics of the disease. 
(3) An increase in the parotid secretory rate, 
which has no clinical consequences but much 
theoretical interest. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



123 



These exocrine glands, different in function and 
in the products they elaborate, are thus affected 
in different ways. The basic defect has not been 
uncovered as yet, but whatever its nature, it ap- 
pears to be genetically transmitted. It is in order 
to explore this problem further with the ultimate 
aim of finding the etiology of the disease and 
therefore a better and more logical approach to 
treatment of the disease that the following in- 
vestigations are planned: 

Chemical Studies of Mucus Structure. Such 
studies may contribute to elucidation of the 
physico-chemical mechanisms involved in the 
higher viscosity of mucus in patients with cystic 
fibrosis and of their tendency to denaturation and 
precipitation which may be one of the major 
pathogenetic factors in this disorder. 

Studies of Intestinal Absorption. Prelimi- 
nary investigations by means of neutral fats and 
fatty acids labeled with I 131 have indicated that 
pancreatic deficiency may not be the only factor 
involved in the intestinal malabsorption of cystic 
fibrosis. These leads are to be pursued further. 
Accurate balance studies of fat and nitrogen 
metabolism have never been performed in the 
recently recognized patients with normal or re- 
duced pancreatic function. Sufficient data are not 
available even in patients with complete pancreatic 
deficiency. In addition, the effects of varying 
dosages of pancreatic extracts have not been care- 
fully assessed. It is planned to carry out further 
studies along these lines. 

Treatment of Pulmonart Involvement. The 
pulmonary involvement dominates the clinical pic- 
ture and determines the fate of the patients with 
cystic fibrosis. Therapeutic trials of antibiotic 
drugs, of enzymes, and physical methods in the 
treatment of this complication are planned. 

PROPOSED STUDIES IN INTESTINAL 
MALABSORPTION IN CHILDREN 

Many of the techniques perfected for the study 
of cystic fibrosis can be applied to further investi- 
gation of other diseases leading to intestinal in- 
sufficiency in children. The recently developed 
fat absorption tests by means of fats tagged with 



I 131 , the oral small intestinal biopsy, and the recog- 
nition of wheat and rye gluten as a noxious 
factor in the diet of many pediatric patients with 
malabsorption offer new tools for investigation of 
this very confused field. 

PROPOSED INVESTIGATIONS IN GLYCOGEN- 
STORAGE DISEASES 

Congenital and usually familial errors of carbo- 
hydrate metabolism lead to a group of disorders 
characterized by accumulation of glycogen in vari- 
ous tissues and organs leading to enlargement and 
dysfunction of the structure involved and, fre- 
quently, to death. 

In recent times by the application of chemical 
techniques several different diseases have been de- 
fined, each one characterized by the absence of an 
enzyme necessary for completion of the carbo- 
hydrate cycle. Much further definition is neces- 
sary as to the metabolic consequences and clinical 
manifestations of these conditions. This repre- 
sents a very dynamic field for investigation and 
other syndromes will undoubtedly be recognized as 
studies are pursued. 

Clinical Endocrinology Branch 

Interest in the Clinical Endocrinology Branch 
has continued to center around carbohydrate 
metabolism and general aspects of the biochem- 
istry of the thyroid gland. The work encompasses 
both clinical and laboratory investigations and in 
many instances studies on patients follow more or 
less directly from results obtained in the labora- 
tory. 

CARBOHYDRATE METABOLISM 

Insulin. A considerable amount of work has 
been done on assays of insulin and insulin inhibi- 
tors in human plasma, the mechanism of insulin 
resistance, and routes of insulin metabolism. As 
a result of modifications introduced in the rat- 
diaphragm assay, it has now been possible to assay 
human serum for insulin-like activity and a value 
in the normal individual of 0.01 milliunits per ml. 
has been found. This is somewhat lower than 
most figures in the literature but is in good ac- 
cord with estimates made from kinetic studies. 



556044—60- 



-10 



124 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The metabolism of insulin in rat-liver perfusion 
experiments and by liver homogenates has been 
investigated. It has been shown that insulin is 
bound in a particulate form in the liver before it 
undergoes proteolysis. The binding of iodo in- 
sulin is competitively inhibited by crystalline in- 
sulin and not by ACTH, growth hormone, or other 
proteins. Hence, this appears to be a specific type 
of bond. At 3° C. it has been shown that binding 
of insulin to subcellular particles proceeds nor- 
mally but proteolysis is markedly slowed, making 
this preparation particularly useful. Interest- 
ingly enough, a liver homogenate which will hy- 
drolyze insulin is unspecifically inhibited by a 
variety of proteins and peptides. These data in 
general demonstrate that an important and early 
step in the metabolism of insulin consists of its 
binding to subcellular particles. 

Work on the effect of insulin on human white- 
blood cells has been commenced. It has been 
shown that leukocytes from both normal and 
diabetic individuals metabolize glucose. The up- 
take of glucose is increased in both cases by in- 
sulin although the formation of carbon dioxide is 
unaffected. 

Carbohydrate Metabolism. The relative impor- 
tance of the hexose monophosphate shunt in the 
whole animal and in intact humans has been 
studied in collaboration with Dr. Berman of the 
Biomathematical Panel. A consistent mathemat- 
ical analysis of the data obtained after admin- 
istration of variously labeled glucose has been 
made. The comparison involves the rate of me- 
tabolism of C 14 from carbon-1 labeled glucose 
versus carbon-6 labeled glucose. It has been 
shown with this technique that in the normal 
human about 10 percent of the glucose is metab- 
olized via the shunt pathway. 

Work has continued in collaboration with Dr. 
Topper on the metabolism of galactose and agents 
which affect it. It has been shown that ethanol 
inhibits the metabolism of galactose in the normal 
individual. In individuals with galactosemia, 
progesterone markedly enhanced the metabolism 
of galactose. Another study utilizing human 
leukocytes has shown marked differences between 
normal and galactosemic leukocytes in their ability 
to metabolize galactose. Preliminary data sug- 
gest that this test is sensitive enough to detect in- 



dividuals with the galactosemic trait but without 
the disease. 



THE THYROID 

Proteins and Proteolysis. A study of the mo- 
lecular properties of thyroglobulin has been ex- 
tended. It has been shown that the denaturation 
of thyroglobulin as observed by its insolubility at 
its isoelectric point obeys first order kinetics. 
Between pH 7 and pH 9, this rate varies but little 
with pH but changes rapidly with temperature 
and an activation energy of 160,000 kilo calories 
per mole has been calculated. Above pH 11, the 
rate increases rapidly with increasing pH and the 
temperature coefficient decreases by about one 
half. The dissociation of thyroglobulin into sub- 
units by alkali which was reported last year was 
postulated to involve a significant activation 
energy. This has been confirmed by the finding 
that an increase in temperature accelerates the 
rates of dissociation and changes the equilibrium 
to favor dissociated products. Evidence so far 
suggests that dissociation precedes denaturation. 
Somewhat surprising has been the observation 
that the dissociated unit (the 12S material) and 
the denatured molecule (8S) both appear to be- 
have as globular proteins when examined by sedi- 
mentation and viscosity. The minimum changes 
in optical rotation attendant upon the production 
of these molecular species tend to confirm this 
picture. 

Investigations have continued on two other spe- 
cies of iodoproteins found in both normal and 
abnormal thyroid tissue. The particulate iodopro- 
tein (Pi) which is particularly high in certain 
strains of transplantable thyroid tumor has been 
further characterized. Contrary to the results re- 
ported last year, the particulate iodoprotein is not 
located in nuclei. Radio-autographs have shown 
that nuclei in general are free of radioiodine and 
differential centrifugation has demonstrated that 
the iodine is located in two different sized par- 
ticles — one about the size of mitochondria and the 
other the size of small microsomes. 

It has now been found that the particulate 
radioiodine can be solubilized by very brief treat- 
ment with either pepsin or trypsin. Fifteen 
minutes at 24° C. solubilizes from 80-90 percent 
of the radioiodine. Chromatography of the solu- 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



125 



bilized material on diethylamine ethyl cellulose 
reveals a single major peak of protein and radio- 
activity and several smaller peaks. Amino anal- 
ysis of the main peak shows a preponderance of 
glutamic and aspartic acids, satisfactorily account- 
ing for an acid isoelectric point. The solubilized 
material when studied in the ultracentrifuge ap- 
peared to be of relatively small molecular weight 
and sedimented slowly. In electrophoresis, the 
material appeared to be homogeneous and with a 
mobility at pH 8.6 similar to that of thyro- 
globulin. The results of analysis of PI for iodoa- 
mino acids have been reported previously as has 
evidence suggesting that it is synthesized inde- 
pendently of thyroglobulin. 

Proteases in the thyroid gland have been the 
object of some recent study. Preliminary experi- 
ments have shown that two proteases with pH op- 
tima of 3.7 and 5.4 can be identified. They have 
been purified by column chromatography about 
500 fold and reasonably well separated. 

Work on thyroxine-binding proteins in animal 
sera has continued in collaboration with Dr. Blum- 
berg of the Arthritis & Rheumatism Branch. The 
thyroxine-binding proteins have been studied with 
starch gel electrophoresis. By this technique, 
four proteins which bind thyroxins have been 
found and utilizing two-dimensional (paper- 
starch gel) electrophoresis, the bands seen in 
starch gel have been identified with the proteins 
separated on paper. It has been shown that pre- 
albumin is identical with the first band on starch 
gel and the a-globulin thyroxine-binding protein 
seen in paper is identical with band 4 on starch 
gel. Studies using ammonium carbonate as a buf- 
fer for paper electrophoresis have shown that the 
capacity of the interalpha thyroxine-binding pro- 
tein (TBG) has a capacity of approximately 0.2 
Hg thyroxine per ml. This is identical with the 
figure previously found when the analysis was 
done in barbital buffer. Prealbumin which is re- 
vealed in ammonium carbonate has been shown to 
have a capacity of approximately 1.5 ng thyroxine 
per ml. Further studies have shown that the 
thyroxine-binding protein which is elevated in 
pregnancy and depressed upon treatment with 
testosterone is identical with TBG as seen in am- 
monium carbonate and with band 4 as identified 
on starch gel electrophoresis. In collaboration 
with Dr. Beierwaltes of the University of Michi- 
gan, an extremely interesting family with congeni- 



tal familial elevation of TBG has been studied. 
The propositus and one of the three children who 
are perfectly euthyroid have serum thyroxine 
levels from two to three times normal. They also 
have TBG levels from four to five times normal. 
These data are consistent with the previous theory 
from this laboratory that, in the interaction of thy- 
roxine and TBG, it is the level of unbound thy- 
roxine which governs the rate of metabolism and 
the effect of this hormone. 

Iodoamino Acids of the Thyroid. An interesting 
reaction of monoiodotyrosine with certain heavy 
metals which results in the formation of a double 
zone on chromatography has been shown. The 
precise mechanism of this reaction is not yet clear. 
Further studies have continued on the synthesis of 
partially and completely hindered analogs of thy- 
roxine. The tertiary butyl quinones I and II re- 
quired in these syntheses and which have never 
previously been described here have been pre- 
pared. In addition, substances III and IV not 
previously known have been synthesized. Both of 
these later two were shown to be biologically active 
in tadpole metamorphosis. 



x 

L 



o=/^ / =0 0== \ 



HO 



HO 




COOH 
CH2CHCOOH 



COOH 
-CH2OHCOOH 



Factors Affecting Thyroid Activity. An ex- 
tremely interesting recent finding in this labora- 
tory has been the demonstration that within 5 
minutes, thyrotropic hormone accelerates the me- 
tabolism of C-l labeled glucose. Under normal 
circumstances in thyroid slices a substantial por- 
tion of glucose appears to be metabolized via the 
hexose monophosphate shunt. TSH appears to in- 
crease the metabolism of glucose via this pathway. 



126 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



This is a specific effect not manifested by a variety 
of other pituitary hormones and not shown by 
TSH on other tissues. The effect of TSH has fur- 
ther been studied in phospholipid synthesis in the 
thyroid. As has been known from previous 
studies, TSH in thyroid slices relatively rapidly 
increases the incorporation of P-32 particularly 
into phosphatidyl inositol. Incorporation of phos- 
phate into phospholipids in cell-free homogenates 
of the thyroid has been difficult to demonstrate in 
more than tracer amounts. However, it seems that 
a-glyceryl phosphate is incorporated in a homog- 
enate system and work is currently under way 
studying these reactions. 

Further work on thyroid biochemistry has been 
concerned with the activity of isolated thyroid 
cells produced by trypsinization of thyroid slices. 
Cells prepared in this manner incorporate iodine 
but do not appear to synthesize thyroglobulin and 
the major iodoprotein made is particulate in na- 
ture. Interestingly enough serum albumin or 
thyroglobulin added to the cells is iodinated. 
Catalase completely inhibits iodination and TSH 
accelerates it. When tyrosine is added to the 
cells, it is iodinated with the formation of free 
MIT. The mechanism involved in these reactions 
is currently under study. 

Metabolic Diseases Branch 

CLINICAL HEMATOLOGICAL RESEARCH 
(STUDIES IN BLOOD DISEASES) 

Biochemistry and Physiology of Initial Stages of 
Blood Coagulation 

The combined work of many investigators has 
thus far indicated that at least five and possibly 
eight different coagulation factors interact during 
the initial stages of blood coagulation to produce 
thromboplastic activity, the activity which con- 
verts prothrombin to thrombin. The numerous 
factors have been identified primarily because each 
factor is responsible for a different congenital 
hemorrhagic disease (the hemophilias and hemo- 
philioid states) yet there is remarkably little in- 
formation concerning the biochemistry of 
thromboplastin formation or of its activity. 

This section's studies have shown that two of the 
factors involved in thromboplastin formation — 



antihemophilic globulin (AHG) and Factor V — 
can be irreversibly inactivated in vitro by agents 
which strongly bind calcium (e.g. ethylenedia- 
mine-tetra-acetic acid (EDTA)) and that it is 
possible to make animals artificially deficient in 
these factors by exchange transfusion with blood 
treated with EDTA. This accomplishment has 
permitted for the first time an evaluation of the 
turnover rates of these factors in normal animals 
with acutely induced deficiency states. Combined 
studies in animals with induced deficiencies and in 
patients with congenital deficiencies have shown 
that the half -life of AHG, of Factor V, and of two 
other thromboplastic factors — plasma thrombo- 
plastin component (PTC) and Factor VII is in 
the order of 6 hours or about 10 times shorter than 
the half-life of other clotting factors not involved 
in thromboplastin activity (prothrombin and 
fibrinogen). These types of in vitro and in vivo 
studies provide information concerning the metab- 
olism and interaction of clotting factors during 
the initial stages of blood coagulation which are 
pertinent to the development of better methods of 
treatment of hemophilioid conditions. 

Stemming from observations (see 1958 report) 
that calcium is an integral part of the AHG and 
Factor V molecules, these NIAMD investigators 
have been measuring the calcium content of differ- 
ent plasma fractions in order to determine whether 
specific clotting factors can be detected and meas- 
ured by means of their calcium content. Prelimi- 
nary results indicate that it may be feasible to 
determine specific deficiencies of clotting factors 
by calcium determination alone and possibly to 
follow changes in the distribution of plasma cal- 
cium in different protein fractions during blood 
coagulation. These studies may prove to be help- 
ful in relating molecular structure of these factors 
to their biochemical activity. 

Clinical Studies of Unusual Coagulation 
Disorders 

Combined clinical and laboratory studies of 
patients with unusal coagulation disorders con- 
tinue to provide further understanding of diseases 
of hemorrhage and thrombosis. The following 
are examples of such studies made during the past 
year: 

1. The mode of inheritance of Factor VII de- 
ficiency was studied by surveying the family of 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



127 



a Navajo Indian patient with this disease. This 
necessitated a field trip to collect blood samples 
and to obtain a careful family protocol on the 
Arizona reservation. Our finding that the disease 
is non-sex-linked and dominant with variable 
penetrance is in agreement with the one other 
genetic study of congenital Factor VII deficiency. 

2. Study of several patients who developed un- 
usual hemorrhagic manifestations while on di- 
cumarol drugs, although adequately controlled 
clinically as determined by prothrombin time 
values, showed that they had, in addition to the 
usual Factor VII and prothrombin deficiency, a 
deficiency of PTC and an abnormality in the 
thromboplastin generation test which suggested 
the lack of an additional factor as well. There 
have been conflicting reports in the literature 
concerning the factors which may occasionally 
become deficient during dicumarol therapy. The 
present studies indicate that PTC as well as 
Factor X are affected and that these factors re- 
main depressed long after prothrombin and Fac- 
tor VII return to normal after discontinuing 
dicumarol. 

3. During the course of evaluating new chemo- 
therapeutic agents, the cancer chemotherapy 
group of the NCI found that the drug 4-amino- 
pyrazolo-pyrimidine (4-APP) produced marked 
prolongation of the prothrombin time in patients 
treated with this compound. Investigations of 
this abnormality by the NIAMD clinical hema- 
tology group showed that 4-APP produced an 
acute transient drop in prothrombin, Factor V, 
and Factor VII concentrations which could not be 
prevented by massive doses of vitamin Ki and 
which could be attributed to hepatocellular dam- 
age. Apart from establishing the precise nature 
of the toxic effect of this drug, these observations 
are of research interest because 4-APP may prove 
to be an excellent agent for producing in labora- 
tory animals controlled specific deficiencies of 
various blood-clotting factors. 

Studies of Prothrombin Conversion 

The transformation of prothrombin to throm- 
bin by thromboplastin (biological activators) has 
been considered to be a relatively simple activa- 
tion of a pro-enzyme. Because little if any 
change in the physical properties of prothrombin 
takes place, it has not been possible to conclude 



from physico-chemical studies that transforma- 
tion of prothrombin into "biothrombin" involves 
formation of other prothrombin derivatives. 
However, kinetic studies of this transformation, 
which were carried out in association with the 
biomathematics group, have demonstrated that 
actually more than one prothombin derivative is 
formed in this step (see 1958 report). Further 
combined experimental and mathematical analy- 
ses of the prothrombin conversion system and of 
its inhibitions by proteolytic enzyme inhibitors 
have resulted in the following basic model for 
the reactions : 

ki k 2 k 3 

Prothrombin ->Derivative-»Derivative— > Thrombin 
\k 8 i + Inhibitor 

Inert compound Derivative-Inhibitor 

Complex 

The kinetic details of thrombin formation and of 
this unique form of competitive inhibition will be 
described in the report of the biomathematics 
group. The fact that such derivatives form ac- 
counts for a number of the puzzling attributes of 
prothrombin conversion in biological systems and 
implies that prothrombin contains several moi- 
eties which may have separate biochemical and 
physiologic functions. 

Study of the Immunology of Blood Cell 
Deficiencies 

'Auto-immunity" (antibodies formed in an in- 
dividual which react with the individual's own 
tissues) has been implicated with increasing fre- 
quency as the basis of diseases involving cellular 
destruction. Some of the most incisive examples 
are hematologic diseases in which a single type of 
circulating blood cell is destroyed by a specific 
antibody which appears to react with one particu- 
lar cellular antigen. Although these hematologic 
immune diseases are relatively well defined, there 
are a number of major questions which have not 
yet been answered: Do antibodies really develop 
against substances which have always been pres- 
ent in the individual? Do some antibodies 
formed against truly foreign antigens attach to 
cells, not by forming a specific antigen-antibody 
complex, but by a more fortuitous process of non- 
specific adsorption on a receptive cell surface? 
Are certain somatic antigens essentially foreign 



128 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



to antibody-forming tissues? Current studies of 
hematologic, auto-immune diseases have been di- 
rected at answering these and similar questions. 

Following the finding by this group (see 1958 
report) that the complex reactions which take 
place between quinidine, antibody, platelets, and 
compliment in quinidine thrombocytopenic pur- 
pura are the same as the reactions which take 
place between stibophen, antibody, red cells, and 
complement in stibophen hemolytic anemia, work 
has been continued with the rare antibody in- 
duced by stibophen in an attempt to resolve the 
question of antigen specificity. Drug purpura 
and drug hemolytic anemia are uniquely suitable 
for such studies because the type, the amount, and 
the rate of antigen- antibody complex formation 
can be controlled by a single factor, the concentra- 
tion of drug. Using specialized radioisotopic 
techniques developed by this laboratory for 
measuring kinetics of antibody complex forma- 
tion with 10~ 9 to 10 -11 M concentrations of anti- 
body, results so far indicate that the first step of 
the overall reaction is the attachment of drug to 
antibody. This is an important finding, for if the 
first step is combination of antibody with drug 
rather than cell with drug, the implications are 
that the cell-drug complex is not the antigen but 
that the antibody-drug complex may be non- 
specifically adsorbed on cell membranes just as 
other non-antibody plasma proteins are adsorbed. 
These studies provide a clearer understanding of 
the basic immunoreactions which can result in 
cellular destruction in vivo and have numerous 
implications in a large group of diseases sus- 
pected of being due to sensitivity reactions. 

Of interest, primarily in the field of immuno- 
chemistry, is the finding that stibophen-antibody- 
cell complexes fix only the second component of 
complement in a hemolytic reaction. The fixation 
of a single complement component (C 2 ) by a 
hemolytic antibody is unique in immunology and 
continued study of this reaction promises to pro- 
vide further information concerning the chemistry 
and significance of the different complement 
components. 

Establishment of a New Syndrome. Studies 
of two patients with an unusual form of idiopa- 
pathic thrombocytopenic purpura (ITP) have 
permitted differentiation of their disease from all 



other types of ITP and definition of a new syn- 
drome. Both patients had a similar atypical 
onset of fulminating thrombocytopenia and were 
unique in that their plasma contained a comple- 
ment-fixing antiplatelet antibody, a finding which 
has never before been described. The patients 
also showed a remarkable peculiarity after re- 
covery in that the platelets which formed in both 
patients would not react with the previously 
isolated antibody of either case, in spite of the 
fact that all normal human platelets and the 
platelets of all animals tested thus far react with 
the antibodies. Further studies have been aimed 
at trying to differentiate the two major possibili- 
ties that the "recovery" platelets are coated with 
some substance (blocking antibody or otherwise) 
which prevents attachment of antibody or that 
a somatic mutation has altered the antigenic prop- 
erties of the platelets. Of special interest and 
value was the finding that this life-threatening 
disease could be cured in a matter of hours by 
exchanging the patient's blood with normal blood 
in order to deplete the body of circulating anti- 
body. The finding for the first time that ITP 
can be caused by a complement-fixing antibody 
is of special significance because up to now there 
has been no proof that ITP is an immunologic 
disease. The establishment of a new thrombo- 
cytopenic syndrome will help to clarify our under- 
standing of the pathogenesis of an obscure group 
of diseases and will provide a rationale for further 
experimental approaches to effective therapy. 

Study of the Normal and Abnormal Physiology 
of Formed Elements of the Blood 

In studies of various patients with refractory 
anemias it has been shown that there is a direct 
correlation between plasma and urinary levels of 
erythropoietine and bone-marrow erythroid cellu- 
larity. The relationship holds even in patients 
whose hypercellular marrow is unable to deliver 
mature red cells to the circulation. Turnover 
studies indicate that in patients with refractory 
anemias the blood-cell-forming elements are being 
replaced at normal or accelerated rates and that 
the marrow responds normally to physiologic 
stimuli. Determination of the factor or factors 
which prevent normal maturation of effective 
oxygen-carrying blood cells will require extensive 
additional study. 



NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 



129 



STUDIES OF HUMAN TOTAL ENERGY 
METABOLISM 

Change in Concept of a Basic Metabolic 
Phenomenon 

Last year's Metabolic Chamber Study of the 
influence of cold environment on the metabolic 
effect of food (specific dynamic effect or SDE) 
produced such an unexpected finding that the 
experiments were repeated in additional young 
male subjects, using instrumentation with im- 
proved sensitivity. The added studies have con- 
firmed the earlier observation that human beings 
differ greatly from dogs in the utilization of 
thermogenesis associated with eating for body- 
heat balance. The studies on dogs, performed 
by Rubner during the classic period of calori- 
metry investigation, had shown that food-induced 
thermogenesis would replace cold-induced thermo- 
genesis and animals fed in the cold did not shiver. 
The current human studies, on the other hand, 
have shown summation of the two types of ther- 
mogenesis; the type from food did not replace 
that from cold. It will be necessary to modify 
the commonly accepted statements on cold-SDE 
thermogenesis interrelationships, which suggest 
that Rubner's work is applicable to all homeother- 
mic species, and indicate inter-species differences. 

Delineation With Fidelity of Moment-to-Moment 
Metabolic Changes Reveals Characteristic Fea- 
tures of Phenomena Obscured by Older Methods 

The unique capacity of the metabolic chamber's 
instrumentation for tracing the patterns of funda- 
mental physiological phenomena has been demon- 
strated in this study of the influence of cold on 
SDE. Not merely the degree of energy expendi- 
ture but the variegated changing form can be out- 
lined by the chamber's system of continuous ex- 
pired gas sampling for minimal changes in oxygen 
and carbon dioxide concentration, in conjunction 
with continuous recording of other physiological 
data such as body temperature at various sites, 
heart rate, etc. Various methods applied in the 
past to human-energy studies have all been based 
on interval sampling of expired air which totally 
obscures moment-to-moment metabolic changes. 
Although cyclic variation in oxygen consumption 
associated with shivering has been suggested in 
previous interval sampling studies, delineation of 



metabolic changes with fidelity which is provided 
by the chamber continuous-sampling procedure 
has made possible the following observations: 
(1) determination of the duration of and interval 
between various bursts of energy expenditure as- 
sociated with shivering, (2) recognition of a sus- 
tained underlying increase in metabolism in the 
cold distinct from the periodic peaks associated 
with gross body shivering, (3) the finding of 
marked interindividual differences in the metabolic 
response to cold, both in lag before initiation and 
in magnitude attained, (4) definition of the dura- 
tion and total amount of metabolic change associ- 
ated with ingestion of food (SDE) and (5) ac- 
curate separation of the metabolic responses due 
to SDE and to cold, and recognition of an altered 
SDE metabolic pattern in the cold as compared 
with its form in a comfortable environment. 

Instrumentation Research 

Initiated for the purpose of altering industrial, 
continuous-flow-gas analyzers specifically for 
metabolic-chamber research (50-fold increase in 
sensitivity desired) , efforts to improve the stability 
and sensitivity of the oxygen analyzer can now 
be considered an instrumentation-research accom- 
plishment. Aided by advice from the NHI Lab- 
oratory of Technical Development, the oxygen 
instrument has been refined to the point where 
0.02 percent changes in oxygen concentration can 
be accurately detected in air streams of 100 liters 
per minute. The carbon-dioxide analyzer was 
modified some time ago and has performed satis- 
factorily over the past 2 years, but steps are under 
way to improve this cell even further. The staff 
of the chamber has also developed a data-handling 
system to deal with the voluminous data gener- 
ated on the strip-chart recorders and to facilitate 
calculations; paper tape from the system will be 
fed to the NIH-IBM computer facility. This 
system has just been installed and is currently 
under test. 

MINERAL METABOLISM STUDIES 

Expansion of the Concept of Altered Bone 
Metabolism in Osteoporosis: Importance of 
Nutritional Factors 

Current isotopic and metabolic-balance studies 
of the patho-physiological processes of mineral 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



metabolism in various bone diseases have been 
emphasizing efforts to determine the pathogenetic 
factors underlying post-menopausal and senile 
osteoporosis. These investigations have progres- 
sed to the point where it seems evident that long- 
accepted concepts are inadequate and a new ap- 
proach is needed. 

The basis for the current studies is as follows : 
Gonadal -hormonal therapy of senile and post- 
menopausal osteoporosis, based on the concept of 
inadequate protein matrix formation, has failed 
to provide a fully satisfactory answer to a dif- 
ficult and increasingly important clinical problem. 
Remineralization has virtually never been seen by 
X-ray following androgen-estrogen therapy. Re- 
cently, the possibility has been raised that the min- 
eral — calcium — has some direct pathogenetic rela- 
tionship to osteoporosis, a condition in which 
bones manifestly contain less calcium than normal. 
Dietary histories in the modest number of patients 
with osteoporosis studied by this Branch have in- 
dicated habitually low calcium intakes in the diet. 
In two large patient series recently reported die- 
tary histories have also indicated significantly 
lower levels of calcium intake in osteoporotic 
patients than in non-osteoporotic individuals. 

First indication that the accepted idea of dimin- 
ished bone formation as the basis for osteoporosis 
might not be correct came from radioisotopic (cal- 
cium-45) studies in our laboratory to determine 
rates of calcium deposition in new bone forma- 
tion. These have revealed apparently normal 
rates in seven patients with osteoporosis, raising 
the possibility that an important mode of de- 
mineralization in this disease is by increased re- 
sorption of bone rather than by diminished bone 
formation; this unexpected finding has been con- 
firmed by a foreign investigator. Metabolic bal- 
ance studies in five patients with osteoporosis 
indicate a positive relationship between calcium 
intake and calcium balance through a wide range 
of intakes (150 to 2,400 mg./day), a relationship 
not previously demonstrated. A widely held con- 



cern that high calcium intakes would lead to more 
frequent development of renal stones has not been 
supported by the metabolic data ; urinary calcium 
levels increased only minimally as the intake was 
raised. These studies have not only indicated 
significant calcium storage by increasing calcium 
intake ; they have also suggested that the calcium 
requirement may vary widely from one individual 
to another and hint that in patients who develop 
osteoporosis the requirement may either be higher 
than for others or that it may be higher than their 
customary intake over many years. Very recent 
balance studies have shown a relative resistance in 
certain patients, as shown by the very high cal- 
cium intake levels required in them to obtain posi- 
tive balance. This suggests that in some patients 
there is a gastro-intestinal absorptive defect for 
calcium and has led to the planning of collabora- 
tive work with the NIAMD gastroenterology 
group to detect the possible presence of subclinical 
and/or selective mineral malabsorption in these 
patients. 

Review of the data acquired to date has led to 
the following formulation : Bearing in mind the 
idea that the skeleton is a great storehouse of 
calcium to be called on when calcium is needed for 
homeostatic control of the miscible calcium pool 
and plasma levels, the concept is suggested that in 
osteoporosis the calcium stores of the skeleton may 
have been gradually drained to compensate for 
losses from the pool by mandatory excretion and 
by bone formation as trabeculae are continually re- 
modeled. The implication is raised that the 
degree to which the skeletal stores have had to be 
called upon has been in proportion to relative in- 
sufficiency of calcium from dietary sources. The 
importance of endocrine hormonal factors is well 
recognized in the pathogenesis of osteoporosis. 
These studies direct attention to dietary mineral 
factors, thus expanding the concept of altered 
bone metabolism in this disease toward recognition 
of a dual set of influences, hormonal and nutri- 
tional. 



NATIONAL INSTITUTE OF MENTAL HEALTH 



AND 



NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES 

AND BLINDNESS 



INTRODUCTION* 

IDEALS PASS INTO GREAT HISTORIC FORCES BY 

EMBODYING THEMSELVES IN INSTITUTIONS 

(Hastings Rashdall, 1895) 

The safeguards of civilization and the purposes 
of civilized man are intimately dependent upon 
ideals that have become embodied in institutions. 
Parliamentary rule, trial by jury, English common 
law, the Federal Constitution, and many agencies 
of our Government (including the Department of 
Health, Education, and Welfare, and the National 
Institutes of Health) are bold and effective insti- 
tutional expressions of ideals. 

Behavior is the outward expression of internal 
values. It follows that fostering socially con- 
structive and adaptive behavior among individu- 
als, institutions, and nations, requires the dis- 
covery, communication, and internalization of 
appropriate values. The magnitude of this task 
seems unimaginable. Perhaps this task looms 
larger than it really should because of our exten- 
sive ignorance and relative neglect of the brain- 
mind as an instrument for social integration. 
Moreover, it is unlikely that we can attain a dif- 
ficult objective without trying ; and it is unlikely 
that we will effectively pursue what is conceived as 
unattainable. 

As I have written in a previous Annual Report 
(1957) : "At the root of the matter are as yet un- 
solved problems relating to the perception of ac- 
tions and of shibboleths, the translation of ideas, 
the momentum of traditional concepts, the adhe- 
sive behavior of groups, the communication of 
ideals and goals. Many scientists have confidence 
that these problems can be solved, given time and 
effort. Our country is presently buying time ; we 
can undoubtedly improve our effort. . . . There 

♦Written by Robert B. Livingston, M.D. 



may be short cuts, but few are evident. We have 
to learn how signals enter the nervous system, how 
they are distorted by concurrent and antecedent 
events, how they relate to mechanisms of reward 
and punishment and emotional expression, how 
learning occurs, and what are the limitations of 
our mnemonic and behavioral response systems. 
These mechanisms have their anatomical, physio- 
logical, chemical, psychological and sociological 
manifestations. What more interesting or im- 
portant labor than to be involved in the unravel- 
ling of these mysteries ?" 

For 200 years western civilization has been pre- 
occupied with the development and distribution 
of energy sources and property. Is it conceiv- 
able that we could now dedicate ourselves more 
conspicuously to fundamental studies concerning 
behavior and particularly social behavior? 

As in previous Annual Reports, the Laboratory 
Chiefs have provided comprehensive statements 
of research progress throughout the year. I have 
attempted here to continue as in the two previous 
Annual Reports an exploration of more general 
scientific issues. These tend to be overlooked in 
the immediacy and seeming urgency of our daily 
undertakings. Yet I believe they are truly per- 
tinent to our ultimate best achievement. 

Raising the level of aspiration and action 
of society requires the widespread internali- 
zation of ideals. Clearly the formulation and 
dissemination of ideals constitute an indis- 
pensable social function. Ideals can shape 
social behavior through their incorporation 
as institutional traditions or other kinds of 
social convention : in this way they can gain 
the force of "unwritten law;" they can be- 
come more powerful than any statutory regu- 
lation. Most social behavior is governed in 
this way, through the popularization of ideals 

131 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



which then operate without need of law or 
administration. Most of our freedom of ac- 
tion and security depends upon conventions 
favoring altruism, faith and mutual trust. 
Yet, even widespread and powerful supports 
for constructive social comportment may be 
too idly held and fall into desuetude : ideals 
tend to "run downhill" and for this reason 
they need to be continuously sought out on a 
higher level and reinforced. 
Ideals are not only important historic forces 
but they evolve historically. Hard-won human 
values are safeguarded and extended mainly 
through the evolution of ideals. For this to occur, 
ideals need to evolve at a rate that will match the 
changing forces of circumstance. Ours is a pe- 
riod when ideals do not seem able to keep pace 
with social, economic, political, and scientific 
changes which appear beyond our understanding 
or control. It may be that every period of his- 
tory has this aspect, yet ours is fortunately the 
only one we have to face. 

Although in "sophisticated" conversation we are 
inclined to deprecate altruism, faith, and mutual 
trust as "unreal" or "impractical," we actually 
live by these ideals. Mankind could not have 
lasted to this risky moment without having devel- 
oped steadfast biological foundations for altruism, 
faith, and mutual trust. These functions are built 
into our chassis, so to speak. They are vital mech- 
anisms which have earned for us our biological 
and social freedom. Coupled with awareness, such 
mechanisms can impel further achievements to en- 
large human dignity and freedom ; it seems neces- 
sary only to encourage a greater awareness of our 
opportunities. Improvement will be measured in 
"little pieces of the striving" in any single act di- 
rected toward the realization of higher ideals. 

The purpose of this essay is to discuss certain 
ideals relating to the pursuit of science, to the in- 
terface between science and society, and to the con- 
tributions which science can make toward the 
development of worthier social purposes and 
means. It is my intention to show that : 

1. The selection of worthier values in a demo- 
cratic society depends upon ideals conceived 
by individuals, especially by individuals pos- 
sessing training and experience in the dis- 
passionate exercise of evaluative skills. In 



recent years this essential process of democ- 
racy has been eroded and given away. 

2. In science there has been an unfortunate re- 
jection of the importance of evaluative judg- 
ments, and of the need for scientists to 
contribute in a professionally broad and re- 
sponsible way to the improvement of social 
purposes and means. Only through an 
effective and disinterested assumption of this 
responsibility by scientists will science itself 
escape from being a toy of technology, pitted 
against all manner of competitive special in- 
terests throughout technology. 

3. From several directions science is revealing 
an ethic based upon scientific rather than 
religious or philosophical grounds. Through 
such findings, science may be enabled to pro- 
vide an increasing power and guidance for 
"life, liberty, and the pursuit of happiness." 

4. Science itself is a valued human enterprise 
which has much to offer society, both apart 
and beyond utility. 

5. What seems most urgently required is first, a 
more successful interface between science and 
society, and second, a greater sense of pro- 
fessional responsibility and probity among 
scientists. This latter calls for the resump- 
tion of an idealistic spirit of craftsmanship, 
something akin perhaps, to the medieval no- 
tion of guild. 

All this requires individual internal actions as 
well as administrative changes relating to condi- 
tions of scientific enterprise. Neither the individ- 
ual internal actions nor the administrative changes 
will suffice alone. They are attainable, together, 
as a natural outgrowth of wider recognition and 
exercise of man's capacity for altruism, faith, and 
mutual trust. 

Sources of Professional Responsibility 

In a democratic community, where can respon- 
sibility be placed for formulating and improving 
ideals and purposes of society and its institutions ? 
Does responsibility lie in the White House? In 
the Cabinet ? In the Congress ? In the Supreme 
Court ? In the communications industry ? In the 
marketplace ? Among the citizenry at large ? 

Improved ways of handling society's problems 
need to be conceived and made available broadly 



MENTAL HEALTH NEUROLOGICAL DISEASES AND BLINDNESS 



133 



throughout society. Through the action of politi- 
cal and social leaders, evolving ideals will influ- 
ence the development and improvement of society's 
purposes and means. Among the most important 
means available are institutions which themselves 
progress through becoming more closely approxi- 
mated to the noble ideals for which they represent 
an embodiment. It is chiefly by this leavening of 
ideals that society can be improved, and chiefly by 
means of institutions that ideals can become "great 
historic forces." Each of us shares responsibility 
for choice of both ends and means in our society. 
Desirably, this responsibility is borne through the 
exercise of individual interpretations, formulated 
as conscientiously and rationally as possible. This 
is essential, by definition, in a democratic com- 
munity. Individual default of conscience in this 
public responsibility is detrimental and morally 
reprehensible. 

In addition to such broad-based individual re- 
sponsibility there is also professional responsi- 
bility, borne variously by lawyers, physicians, 
teachers, scientists, civil servants, and others ; pro- 
fessional responsibility which is far heavier than 
that borne by the citizens-at-large. This addi- 
tional responsibility grows out of the professional 
skills and experience of the individual. Thus, as 
scientists, we bear a special social responsibility 
because of our first-hand knowledge of the nature 
a,nd potentialities of science. This makes us re- 
sponsible not only for the excellence of investiga- 
tive work for which we are more or less directly 
responsible, but also for contributions of pro- 
fessional insight and effort toward achieving a 
worthier destiny for our social and institutional 
environments. 

Our responsibility in this more inclusive 
professional sense derives quite naturally from 
the facts: (1) that our own professional 
destiny is intimately bound up with the 
achievements of our immediate professional 
community, (2) that this group in turn has 
the capacity to contribute more effectively to 
the achievements of our society, and (3) that 
our responsibility for a share in making this 
go well in its entirety is neglected at our own 
peril, individually, institutionally and as a 
society. 
Somehow organizational and societal bigness 
has induced a psychological dwarfing of the con- 



ception of the only proper role of the individual 
in a democracy. The town-meeting ideal has been 
lost to some degree, and individual responsibility, 
especially individual professional responsibility, 
has been eroded and given away. Responsibility 
has been eroded insofar as individuals and insti- 
tutions reject or fail to allow for it. Rejection 
is of course encouraged by those whose ambition 
favors their own limited interest. Failure to allow 
for responsibility has two principal origins: one, 
a lack of faith in the willingness or capacity of 
individuals to bear such responsibility ; the other, 
an unwillingness to cope with confusions that oc- 
cur with the widespread exercise of responsibility. 
Responsibility has been given away insofar as 
those by whom it should be exercised have not 
conceived the need for it or because they prefer 
responsibility that is limted strictly to their own 
immediate work, or because they accept a view 
of themselves as helpless cogs, too ineffectual or 
too inadequately informed to be able to exert an 
effect on the massive and supposedly inflexible 
institutions of society. 

To the extent that individual responsibility and 
especially individual professional responsibility 
is precluded or avoided, our institutions and 
society are made to depend on undemocratic pro- 
cedures. This is perhaps not disadvantageous in 
itself in the short view, and under broadly re- 
sponsible and objective leadership, but it inevi- 
tably entails two further substantial losses, both 
of which bear importantly upon our ultimate insti- 
tutional and social accomplishment. First, there 
is a loss of the many conscientious and responsible 
intellectual contributions which otherwise could 
have been made toward a more desirable destiny. 
Second, there is a subtler but more influential 
loss of group identification and motivation which 
otherwise derives from the sharing of social re- 
sponsibility. Finally, as John Stuart Mill said, 
an institution or society "which dwarfs its men, 
in order that they may be more docile instru- 
ments in its hands even for beneficial purposes, 
will find that with small men no great thing can 
really be accomplished." 

Professional Evaluations Indispensable to 
Science and Society 

It is often supposed that scientists deal ex- 
clusively with facts, and that they eliminate from 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



their deliberations considerations concerning 
values. It is supposed that facts can be assembled 
and systematized in relation to other facts, where- 
upon out of an examination of such relations will 
emerge general "laws of nature." This is a mis- 
taken view. Actions stemming from this false 
supposition may be devastating. Scientists have 
been blocked or dismissed from responsible posi- 
tions, in part at least on the assumption that their 
reasoning can and should exist in isolation from 
considerations of value. This primitive notion 
that scientists should be professionally obligated 
to reject value discriminations, or that they are 
ill-fitted by their scientific experience to make 
value discriminations, represents a completely in- 
adequate conception of the scope and method of 
science. I believe that the practice of science 
requires certain value analyses that are indispen- 
sable to science and society. These include (1) 
evaluations intrinsic to the scientific method; (2) 
evaluations covering professional excellence in 
science, and (3) evaluations relating science to 
society. 

Evaluations in Science. It is true that in the 
factual stage of inquiry scientists try to character- 
ize their observations in a form as free as possible 
from personal bias and opinion; this is an ideal 
toward which all scientists strive. But facts, even 
when ideally established, are only bricks from 
which the structure of science is developed. By 
themselves, facts tend to be uninteresting. The 
really significant features of science are established 
from facts which are given meaning, i.e., value, 
through conceptual thinking. 

From start to finish of any scientific problem, 
scientists are engaged in value discriminations, in 
committing themselves to choices which severely 
delimit whatever may be the ultimate value of 
their scientific accomplishment. The selection of 
a problem to study, choice of methods, develop- 
ment of conceptual and technical operational defi- 
nitions, attempts to isolate facts from artifacts and 
from underlying assumptions, selection of those 
facts presumed to be objectively meaningful, inter- 
pretation of the factual data (which are inevitably 
compounded of theoretical interpretations and 
sense perceptions) , and the representation of these 
data and interpretations for the purposes of mean- 
ingful communication — each step in scientific ac- 



complishment requires value discriminations of a 
high order. A differential capacity for handling 
these difficult discriminations is the principal dis- 
tinction between truly great and lesser scientists. 
It can readily be conceded that only scientists 
are professionally qualified to make judgments 
concerning values that are intrinsic to science. 
Yet it is seldom remembered that the profession of 
science demands a continuing exercise of vaT/ue 
discriminations and provides disciplined experi- 
ence in making such evaluations. 

Evaluations of Scientists. All of science and 
technology depends upon a small but indispens- 
able population of creative scientists. Such men 
are professionally disciplined to deal with pe- 
culiar instrumental devices, with abstract thought 
at the limits of conception, and with certain 
general principles which guide their intellectual 
progression. What a scientist sees with his in- 
struments and what he interprets from these 
revelations is by no means obvious. As I have 
written elsewhere, "A more adequate understand- 
ing of nature cannot be achieved in the abstract; 
it must be brought about through the considera- 
tion of materials with which the scientist is al- 
ready familiar. Even the most gifted and 
energetic person must have achieved a certain 
mastery in the field of his pretended accomplish- 
ments. He must have a keen sense of what needs 
to be done to solve a given problem and a suffi- 
cient skill to do that. . . . Important scientific 
achievements thus seem to depend upon the fruit- 
ful combination of a group of essentially positive 
factors; some of these relate to the competence, 
self-discipline, and nimble imaginativeness of the 
scientist himself, and others concern his surround- 
ings. Eesearch in laboratories of the Federal 
Government will surely progress in the sense of 
advancing the frontier. And the rate of advance- 
ment may be speeded up somewhat by administra- 
tive hustling or by providing additional money 
or personnel in a given field. But saltatory ad- 
vancement of concepts — the kinds of change in 
point-of-view that may alter the entire character 
and direction of scientific pursuit, the kinds of ad- 
vancement that may cut short years of striving — 
these are not likely to occur except where 
circumstances are especially favorable for crea- 
tivity. In the long run, the reputation and credit 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



135 



of any laboratory will depend upon a few ad- 
vances of this sort far more than upon the 
extension of studies that now seem entirely 
familiar." 

The creation of worthier new concepts in sci- 
ence is impossible without intellectual noncon- 
formity. What is considered to be "logical 
reasoning" evolves as a delayed consequence of 
scientific achievement; thus the steps in the 
formation of a new concept not only seem alien 
and eccentric and in conflict with common sense, 
but they frequently seem "illogical." It must be 
remembered that any concept is a "freely chosen 
convention" and nothing more: it is yielded 
through intuitive and nonlogical mental processes 
which are not under any satisfactory degree of 
voluntary control. Abstract ideas involved in the 
creation of a new concept need to be "played 
with" imaginatively, often over a period of years, 
before a truly new level of understanding is 
achieved. It is only after a new concept has been 
clearly differentiated that the logical processes of 
science and the disciplined testing against sense 
experience can be pursued. 

The history of the growth of scientific con- 
cepts makes obvious a primary requirement in 
scientists of a high-level capacity for conceptual 
thinking coupled with a capacity not to hold any 
concept too dearly. Widespread acceptance of a 
new level of understanding in science is achieved 
through the examination of evidence that is made 
as free as possible from personal appeal, coercion, 
or "fashion" of thinking, and without recourse to 
authority external to the body of science. As a 
system of thought science is practically unique in 
not being imposed by coercion or persuasion, and 
in not being destroyed when found internally 
inconsistent; paradoxically, science becomes 
stronger and more coherent as its limited views 
are made manifest. The search for a Scientific 
Truth (which can never be realized) becomes 
ever more powerful as error is discovered in lesser 
"scientific truths." 

Science, in contrast with many other callings, 
naturally creates a zeal for integrity ; a lie in sci- 
ence cannot persist, for it will be found out 
through the continuing activities of science. Sci- 
entists are professionally indoctrinated to the 
practice of probity. They fail in this regard only 



insofar as they are persuaded to abandon their 
professional role in society. 

Scientists alone are adequately qualified to 
evaluate a fellow scientist and his scientific per- 
formance. Since scientific accomplishments of 
high quality are the sine qua non for the ex- 
istence of a scientific establishment, a code for the 
selection and promotion of scientists must be 
based upon scientific considerations applied by 
scientists knowledgeable concerning both the in- 
dividual and his field of learning. 

The profession of scientists is uniquely quali- 
fied to provide those discriminations which will 
foster genuine scientific excellence. They alone 
can prevent freedom, which creativity requires, 
from being used as shelter for inefficiency, super- 
ficiality or uncritical partisanship. Scientists are 
well aware that if their own profession does not 
provide these discriminative evaluations, they 
will have to be made by others who may lack the 
necessary qualifications. If this comes about 
(through casualness or default of the scientists 
concerned, or by direction of persons unfamiliar 
with the values and conditions essential to profes- 
sional excellence), the resulting actions are cer- 
tain to breed suspicion and controversy that will 
be deeply injurious to the internal order and to 
the external standing of the institution. 

Evaluations or Science in Relation to Society. 
Science consists of a collection of information, a 
body of theory, and a methodology. All the dis- 
ciplines of science share a dedication to certain 
general principles of inquiry and evidence: this 
forms the only basis for the unity of science. 
Science is one of the few creative, truly progres- 
sive, intellectual activities. Theoretical notions 
tested and found valid are of use in the pursuit of 
further understanding. It is this progressiveness 
which gives science much of its power. A further 
source of power derives from the scientist's in- 
ternal discipline to seek simpler and more general 
expressions which can account for the vast 
schemes of nature. 

Since World War I, science has become domi- 
nant in generating and directing the development 
of technology. In earlier years, the relations be- 
tween science and empirical discovery were spo- 
radic, with practice influencing theory more often 
than the reverse. Since World War II, science 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



and technology have been more and more lumped 
together. There is now developing a widespread 
concern within the Executive and Legislative 
Branches of the Government regarding the extent 
of tolerance to be allowed for the "tyranny" of 
which science and technology seem to be capable. 
The question of how, i.e., by whom and by what 
criteria science is to be evaluated, is acute as well 
as important. 

Several serious problems need to be ad- 
dressed : How does science need to be distin- 
guished from technology in terms of both its 
planning and realization? How can the in- 
tellectual content and power of the educa- 
tional and research activities of science be 
strengthened? (How can the mutual inter- 
dependence of science and the humanities be 
more fully recognized and made effectual?) 
What branches of science need encouragement 
for the immediate and more distant scientific 
and technological advantages of society? 
(How can scientists best participate in the 
social value determinations this requires?) 
How can program developments in science be 
generated and encouraged more in accord- 
ance with professional scientific rather than 
simply political and economic conceptions of 
need and of research potential ? In a science 
mart of limited resources, how can the tend- 
ency for competitive over-justification, for 
"tyrannizing with facts" by scientists be dis- 
couraged? (How can such forthright and 
natural traits as professional congruity and 
candidness be given greater encouragement ? ) 
Few of the answers recommended for these 
problems have been put to any test. The prob- 
lems themselves are not diminishing; they are 
getting worse as public demands and needs are ex- 
panding, and as competition beyond the control 
of our society is exerting an avalanche of pres- 
sures on our technology. These problems have 
been addressed in different ways and with per- 
haps greater degrees of success in some other demo- 
cratic communities. The United States clearly has 
no monopoly on creativity in science and no 
surety for the best ways to utilize such talent in 
improving, safeguarding, and attaining a worthier 
destiny for mankind. We need speedily to bring 
these issues to more objective analysis and to work 
out ways for improving the interdependent work- 



ing relations between science and society. To this 
end, there is an imperative need to stop eroding 
and giving away professional responsibility which 
belongs to scientists. 

Power and wealth are actively sought; tech- 
nology yields power and wealth; technology is 
dependent upon science. Yet because of vast dis- 
crepancies in their relative costs, there is danger 
that science, whenever lumped together with tech- 
nology, will be conceived as riding on the coattails 
of technology rather than the other way around. 

We need to make a fresh analysis of the 
role of Governmental institutions bearing di- 
rectly or indirectly on science: what sufficed 
for a realization of ideals for democratic Gov- 
ernment in the late 18th and 19th centuries, 
when science was a negligible factor in the 
health, welfare and defense of society, may 
require revision now when science has become 
so prominent and indispensable. It is manda- 
tory that any institutional revisions be per- 
formed with conscious deliberation and wis- 
dom instead of crisis to crisis improvisation. 
Our Government will enjoy wisdom in its 
councils to the degree that it can understand 
and foster wisdom and can distinguish this 
from the cacophony of limited- interest ap- 
peals. Decision making, in areas relating 
science to society, should be as carefully ob- 
jectified and deliberated as decisions and in- 
terpretations in the field of law by our 
Supreme Court. 
It is evident that scientists are needed not only 
for the evaluations of science, of scientists, and of 
scientific performance, but also, for the difficult 
judgmental evaluations relating science to society. 
They are needed as full and responsible par- 
ticipants throughout the decision making processes 
involved in the conception and realization of so- 
ciety's goals. Although all of this seems patently 
true, when it comes to practice there are obstacles. 
Some of these arise out of the tendency to depre- 
cate the scientists' training and capacity for mak- 
ing evaluative judgments. A second obstacle 
results from the expectation that scientific values 
are to be measured according to marketplace 
values ; that what is scientifically "good" or "bad" 
is determined, as are so many other social values, 
by some kind of scale of popularity, through per- 
sonal suasion, coercion, or appeal to external au- 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



137 



thority. Some scientists may be persuaded into a 
degree of conformity to this expectation, especially 
when it is held rather uniformly by those con- 
trolling the supports of science. Kesponding to 
such an expectation, scientists try to express the 
goals of their scientific endeavors entirely in terms 
of technological and marketplace considerations. 
This in itself is a principal obstacle to the cultiva- 
tion of high quality science. It is a barrier to 
understanding the nature and scope of science by 
a wider public. 

The degree to which scientists make use of "a 
tyranny of facts" or other limited-interest tech- 
niques which violate professional probity is a 
measure of their failure to qualify properly for 
bearing their valid professional responsibilities. 
It is not so much that the facts and concepts of 
science need translating for the public as that the 
purposes and systems of value of science need 
translating. 

The Evolution of Human Values 

Evolution, as popularly understood, emphasizes 
conflict as the principal fulcrum around which 
evolutionary progress takes place. It deempha- 
sizes altruism and cooperation as contributing im- 
portantly to evolution. The popular derivations 
from the teachings of Darwin and, indeed, of 
Marx and Freud as well, give us only half of our 
nature. Conflict cannot be put aside altogether, 
but as an instrument for evolution, conflict alone is 
like the odd half of a pair of scissors. An em- 
phasis on conflict as the basis for individual or 
collective evolution reveals only half our oppor- 
tunity, half our capability, and half our responsi- 
bility. 

How this notion of the significance of conflict 
can have become so widely accepted in the face of 
commonplace evidence to the contrary, how it can 
have magnified the acceptance of conflict as a 
way to the solution of problems, and how it can 
have secured the social acceptance of conflict to 
the degree that it has is beyond my understanding. 
It is not that Darwin, Marx, or Freud accom- 
plished this directly, because popular conceptions 
embrace only fragmentary parts of their contri- 
butions. It has been simpler, and hence more 
popular, to believe that evolution proceeds pre- 
dominantly through success or failure in conflict 



situations, and that an individual succeeds or 
fails according to his natural endowments, in 
which his potentialities for conflict are of para- 
mount importance / that great social forces stem 
from conflict in which poioer, aggressiveness, and 
wealth are principal determinants ; that the indi- 
vidual is merely a moving atomy of conflicts, 
each being the victim of instinctual drives, chiefly 
of a gross and disagreeable nature. 

A more valid thesis, I believe, is that altruism, 
faith, and mutual trust are built into our behavior 
just as surely as are the mechanisms for aggres- 
sion and conflict. Each of these systems repre- 
sents a vital force which has developed progres- 
sively throughout phylogeny. Each has played 
a central role in determination of freedom as well 
as survival and creative evolution of biological 
organizations. 

Plants, which must remain relatively fixed 
in relation to their environments, are capable 
of living off fairly homogeneously distrib- 
uted raw chemicals. Biological discrimina- 
tions upon which plants depend are concerned 
with relatively elementary chemical and 
physical factor. Animals, on the other hand, 
depend upon partly organized chemical sub- 
stances which are heterogeneously distrib- 
uted and which they must actively seek out. 
Animals must be able to discriminate objects 
in their environment which may provide suit- 
able energy sources, and to secure them for 
their own and their progeny's use. Animals 
are characteristically mobile, built for action. 
The evolving nervous system has from the 
beginning been a system that is both selective 
and directive. Higher animals have more 
complex systems for discrimination and 
action, and a greater capacity to learn new 
discriminations and new actions. 

The ability to discriminate is intimately re- 
lated to what we call appetite, feeling and 
emotion, and also with mechanisms concerned 
in the direction of action. Behavior is gen- 
erated by motivations which in turn are 
shaped by biological systems of value, 
whether these are consciously manifest or not. 
Built into such differentiating-action-generat- 
ing systems are vitally essential mechanisms 
for value discriminations affecting preserva- 
tion of individuals and the species. Survival 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



and evolution could not have gone very far 
in the creation of complex forms of life with- 
out having biological foundations for altru- 
ism, faith and mutual trust, in addition to 
mechanisms for combat. Cooperation and 
faith in some degree are absolutely essential 
for the reproduction and survival of most off- 
spring, and are admittedly very far-reaching 
in yielding internal biological satisfactions. 
It must be remembered that neither aggression 
nor altruism requires deliberate conscious par- 
ticipation, even in those organisms capable of 
consciousness. 

We act, and live, by faith: faith in ourselves; 
faith in the consistency of nature; faith in each 
other. Every perception and every overt act is 
based on faith. Action follows the state of the 
nervous system whether it be so-called "spon- 
taneous" action, reflex action or action of the 
"will." The state of the nervous system (the 
brain-mind) is variously called an image, an idea, 
a feeling, an emotion, or a judgment which in turn 
is based upon comparative evaluations of various 
•images, ideas, feelings, etc., built up and stored 
during previous experiences of the species and of 
the individual. Knowledge of the outside world 
(and of ourselves) largely evolves out of a cumu- 
lative experience which begins with our own 
"spontaneous" actions. Deliberated decisions — 
even decisions based on strong feelings of "will" — 
are largely founded on systems of experiential 
consistency and the projected faith derived from 
that. 

To be determined by one's own nature is to be 
free. An educated man is said to be one able to 
foresee the consequences of his actions in the 
widest possible totality of their relationships 
(Kermit Eby, 1951). A wise man is most fully 
self-aware. He is sensitively empathic regarding 
the possible consequences of his actions considered 
in the widest context and in the longest view. A 
wise and resolute person has a store of stable and 
worthy ends, patience, and a style for engagement 
in action. Consciousness (and the contributions 
of education and wisdom to a conscious and reso- 
lute person) provides the fullest opportunity for 
the development and exercise of capacities for 
altruism, faith and mutual trust, which none- 
theless remain just as much natural and vital 



mechanisms as are the most primitive acts of co- 
operation observed in lower animals. 

Freedom to act purposefully with intelligent 
foresight of the probable consequences of action 
stands as moral freedom when the social con- 
sequences of the action are taken into account. 
These actions are also both natural and vital. 
They relate importantly to survival and embody 
the realization of vital satisfactions. "Man's 
capacity for intelligently directed self-develop- 
ment confers upon him the ability to determine 
the pattern of his culture and so to shape the 
course of human evolution in directions of his own 
choice. This ability, which no other animals 
have, is man's most distinctive characteristic, and 
it is perhaps the most significant fact known to 
science"" (C. Judson Herrick, 1956). 

The brain-mind is an evolutionary tool like 
teeth and claws, but we can expect from it a 
much more creative performance. The brain- 
mind of man is highly developed with respect 
to its capacity for discrimination among ob- 
jects of the environment in favor of suitable 
energy sources. The present uneven distribu- 
tion of wealth and power throughout the 
world is largely due to the purposeful use of 
the brain-mind according to the cumulative 
methodology of science. Yet garnering and 
exploiting energy sources is not the greatest 
nor loftiest purpose of mankind. With all 
that the brain-mind has accomplished, it is 
still a very incompletely exploited instrument 
for contributing to the extension of freedom 
and to the further encouragement of man's 
natural capacities for altruism, faith and mu- 
tual trust. 
For many years psychologists, sociologists, and 
social anthropologists have studied human capa- 
bilities and limitations in this regard. Recently 
Dr. Marion Yarrow and Dr. John Campbell have 
shown that children brought into newly formed 
children's groups display differences in initial re- 
lations which are predictive of their later social 
effectiveness within that group. Children who on 
first acquaintance find other children attractive 
come to be the best liked and most effective mem- 
bers of the group in terms of acceptance and in- 
fluence. Children who are initially aggressive or 
angry in their reactions become typed in this char- 
acterization and are impaired in their perception 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



139 



of others as well as being less effective in their 
later interactions within the group. More recently 
has appeared a growing insight into what may be 
the internal biological mechanisms underlying 
altruism, faith, and mutual trust. The work of 
Dr. Paul MacLean is particularly significant in 
this regard. He has localized two separate brain 
regions, one of which concerns behaviors leading 
to preservation of the individual and the other of 
which concerns behaviors leading to preservation 
of the species. Each of these regions incorporates 
mechanisms both for cooperation and for combat. 
In actuality, several disciplines basic to neurology 
and psychiatry are contributing to a system of 
ethics that can be developed without dogma and 
entirely on scientific grounds. 

Scientific contributions relating to conscious- 
ness, perception, appetite, emotion, learning, mem- 
ory, motivation, value discrimination, decision 
making, social awareness and responsiveness, and 
will are all pertinent to considerations of what 
we have to deal with in human nature. They are 
profoundly important in relation (as above) to 
"the perception of actions and of shibboleth, the 
translation of ideas, the momentum of traditional 
concepts, the adhesive behavior of groups, the 
communication of ideals and goals." At the very 
least, contemporary research indicates that human 
nature may be considered from a positive as well 
as from the more traditionally negative point of 
view. Such contributions, being cumulative, will 
provide a continuing improvement of our under- 
standing of the potentialities of human behavior. 
These fields of science, although late to mature, 
portend to contribute more importantly than any 
other intellectual enterprise of man to his ultimate 
fulfillment in "life, liberty and the pursuit of 
happiness." 

Science as a Human Value 

Science itself has evolved as a valued human 
enterprise. Emphasis has been greatest in this 
country, and especially in recent times, upon 
science as valuable from a predominantly utilitar- 
ian point of view. Science has proven so useful in 
finding and exploiting suitable energy sources, 
that its contributions through technology to the 
standard of living is taken by many to be the chief 
social value of science. Yet those who have heard 
the "beep" of an earth satellite or have seen one 



crossing the sky, have experienced an inevitable 
cultural thrill through their own perceptual con- 
firmation that man can do such a thing. This re- 
sponse carries with it the further recognition that 
the world will never be the same. Science in this 
way fulfills a part of man's innate curiosity. Al- 
most all of astronomy and astrophysics, most of 
the earth sciences, and biomedical sciences in par- 
ticular, contribute to fulfilling the innate desire 
of man to know, to understand, to comprehend 
the universe and himself. Scientific discovery 
ultimately has a cultural impact upon the philoso- 
phy of thought and upon the vitality of ideas. 
Science stresses that the individual, community, 
and the universe itself, is always in a process of 
becoming, and that none of this transaction can be 
made to stand still. There is a certain anti-inertial 
force provided society through scientific enter- 
prise. Science debunks authority; it emphasizes 
the intrinsically creative aspect of man's own life 
and his capacity to create increasing freedom 
within the total domain of organic and inorganic 
evolution. In many ways, science provides useful 
implements for cultural development. 

Science is not a body of dogma: It is a way 
of life. The requirements of the creative process 
impose self -discipline and intellectual integrity. 
In the pursuit of science only that which can be 
communicated and sustained by others is retained 
and dignified as part of the organized knowledge 
of science. The process of creating new con- 
cepts requires maximum freedom. Progressively 
less freedom is needed for the exploitation of 
available concepts, hence, for development and 
technology. The need for freedom, freedom in 
thinking, freedom in discussion, freedom to 
demonstrate the true nature of man and his 
society, freedom of publication, all required by 
science, is a further contribution to the strength 
of freedom throughout society. Acceptance of the 
spirit and methodology of science by society's 
leaders assists society in adapting to new situa- 
tions without the kinds of fear which have 
attended drastic changes in the past. 

Culture is affected by the challenge of the 
adventure of science, of the frontiers to be sur- 
passed, of the beckoning effect of the unknown. 
Science provides concepts of enormous intellec- 
tual satisfaction, enrichment and entertainment. 
Science contributes to the discipline of a culti- 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



vated society and to the inspiration of its youth. 
Thorstein Veblen wrote in 1906, "In myth-making, 
folklore, and occult symbolism many of the lower 
barbarians have achieved things beyond what the 
latter-day priests and poets know how to propose. 
In political finesse, as well as in unreasoning, 
brute loyalty, more than one of the ancient peoples 
gives evidence of a capacity to which no modern 
civilized nation may aspire. To modern civilized 
men, especially in their intervals of sober reflec- 
tion, all these things that distinguish the barbari- 
an civilizations seem of dubious value — futile in 
comparison with the achievements of science. 
They dwindle in men's esteem as time passes. 
This is the one secure holding-ground of latter- 
day convictions, that 'the increase and diffusion 
of knowledge among men' is indefeasibly right 
and good. When seen in such perspective as will 
clear it of the trivial perplexities of work day 
life, this proposition is not questioned within the 
horizon of Western culture, and no other cultural 
ideal holds a similar unquestioned place in the 
convictions of civilized mankind." 



Toward an Ideal Destiny 

Cultural Differences Affecting Science in 
Relation to Society. A research enterprise that 
depends upon the patronage of a democratic soci- 
ety depends upon a relatively broad understanding 
throughout that society of the values of science 
and of the conditions under which science can 
flourish or will languish. An obstacle to such 
understanding is that society is made up of many 
different cultural groups, each of which has its 
own set of values and conception of the conditions 
necessary to its own kind of enterprise. Examples 
of such groups are schoolboys, preachers, artists, 
salesmen, teachers, thieves, sailors, playwrights, 
physicians, policemen, pilots, businessmen, factory 
workers, television sponsors, miners, research sci- 
entists, bankers, and soldiers. In general, there 
exist only limited cross-group familiarities, al- 
though schoolboys, as a group, study teachers, and 
vice versa; thieves study policemen, etc. An in- 
dividual in one group is likely to judge the actions 
of members of any other group according to his 
own code: indeed, he may know no other. The 
simplest translations are between groups whose 



values, actions, and conditions of work are known 
to each other through continuing interaction. Yet 
difficult translations may be required between 
groups whose superficial familiarity with each 
other may blind them to fundamental underlying 
differences. 

The predominant system of values and con- 
ception of working conditions in our society 
(at this time) relates to the marketplace. By 
and large, the leaders of our society under- 
stand the values and conditions relating to 
successful business and political enterprise. 
This is the code which is also most often 
publicly interpreted by the communications 
industry. This code is therefore the common- 
place and primary cultural reference by which 
actions are interpreted. For these reasons it 
is quite understandable, although regrettable, 
that a research enterprise is likely to be eval- 
uated according to marketplace standards. 

A further general feature of the action 
interface among different cultural groups is 
that the 'predominant group not only evalu- 
ates the actions of other groups in the light 
of its own system of values, out that it ac- 
tively exerts pressures to compel conformity 
of action in accordance toith that same sys- 
tem. Anything else would seem "alien" and 
"illogical," if not "improper," according to 
the code of the predominant group. This 
tendency is entirely natural, and is equally 
unreasoned. As an example: pressures are 
exerted, directly and indirectly, by the pre- 
dominant group of righthanded persons to 
disguise or eliminate lef ^handedness ; at the 
very least, to require lefthanded persons to 
adapt themselves to the way in which hands 
are to be shaken, tables set, doors opened, 
faucets turned, writing desks arranged, etc. 
These two general facts of cultural interaction, 
compelling judgment and conformity to a foreign 
code, have a powerful influence upon the action 
interface existing between scientists engaged in 
research and the patrons of science. Business and 
politics are fields of competition, as is science. 
Yet, the basis for the competition is fundamentally 
different. Business and politics are mainly for 
the purposes of social service, social power and 
social control. To a large degree the marketplace 
and public opinion determine what is correct, 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



141 



what measures success, and what standards of 
conduct must be met. Most trustees of universities 
and managers of business as well as most members 
of the Congress and Federal Executives, are men 
with extensive experience in the professions of 
business, law and politics, but little or none in 
the direct pursuit of science. They are, therefore, 
in general, prepared to predict the social useful- 
ness of a product, to estimate the popularity of 
a public policy, and to evaluate the risks and 
costs of an economic venture. They are culturally 
bound, perforce of their own code and previous 
experience, to evaluate a scientific enterprise in 
accordance with such terms. Only very rarely 
are such individuals experienced in judging cre- 
ative scientific endeavor, scientific concepts, or the 
conditions essential to professional excellence in 
science. Moreover, because technology requires 
conditions that are easier to appreciate according 
to marketplace standards, and because science and 
technology are often lumped together, mistaken 
judgments arising out of a confusion of these two 
activities are unfortunately often reinforced in an 
individuals experience, to the obvious detriment 
of science. 

Attracting the Ablest Scientists to an Organ- 
ization. There is no substitue for setting the 
highest standards, and for providing the greatest 
attraction possible, for key scientific personnel. 
A relatively few top-quality individual scientists 
can provide an aura of excellence for the entire 
organization that will conclusively ensure future 
recruitment and retention. Such individuals will 
illustrate the creative process, the internal self- 
discipline, the professional competence, and the 
intellectual devotion required by science. They 
will live out the satisfactions which derive from 
intellectual pursuits and set the intellectual and 
experimental pace for the scientific community, 
according to their own lights. They will ensure 
the establishment of traditions most suitable for 
individual professional development and achieve- 
ment. Other scientists, whether beginners or es- 
tablished investigators, will draw pride from 
association with these individuals and their pro- 
fessional accomplishments. 

It is clear that without substantial evidence of 
encouraging creative accomplishment and of pro- 
viding creative individuals positions where they 



can accomplish their maximum, an organization 
is bound to languish as a scientific institution. 
Without substantial professional recognition both 
within and outside of the organization, no other 
recognition is meaningful. It might be urged that 
since only a relatively few individuals within an 
organization are likely to be highly creative, it 
is not necessary to indulge in developing a truly 
creative environment: this is a ruinous miscon- 
ception. If an institution is unable, for whatever 
reasons, to attract and to keep the indispensable 
(even though small) fraction of highly creative 
scientists upon whom its professional reputation 
depends, it can lose nothing not already lost. 

When even a few highly creative scientists find 
that a given institution is best from the point of 
view of their individual professional development 
and accomplishment, then there are few obstacles 
to the administration of that organization: 

(1) Eecruitment and retention of top-level 
scientists is made easy. 

(2) A scientist who must leave the or- 
ganization because a position can no longer 
be made available to him, leaves with a sense 
of pride in his professional experience and 
association with the organization ; on the out- 
side, he is a knowledgeable advocate for its 
scientific program and its professional 
support. 

(3) Internal professional ideals to seek 
greater freedom, dignity and responsibility 
for the individual scientist, tend in a self- 
controlling, group-correcting way to elevate 
the standards of excellence of scientific per- 
formance entirely in the absence of admin- 
istrative intervention. 

(4) The stature and license of admin- 
istration as representative of this respected 
community of scholars become automatically 
enhanced. 

(5) Advocacy for support of the organ- 
ization's program becomes more objectively 
scientific and less political in character; this, 
in turn, has a strong and favorable effect 
back upon the professional reputation of the 
organization. 

(6) The value system of individual sci- 
entists becomes more closely identified with 
the professional excellence of the organiza- 
tion and less concerned with emoluments ; yet 



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ANNUAL REVIEW OP INTRAMURAL RESEARCH 



at the same time improved emoluments be- 
come even more evidently deserved and easier 
to justify and to acquire. 
The ablest scientists seek an environment where 
limitations to their accomplishing important in- 
tellectual work will be mostly internal, where few 
limitations can be assigned to the environment. 
They seek a setting where they can have the 
greatest scope and freedom for both the pre- 
logical and logical steps in their scientific work. 
These issues seem intangible, but their implica- 
tions reach into every aspect of daily life: does 
the organization buy the scientist's time and then 
give it back to him to employ according to his 
own conception of time's most fruitful utiliza- 
tion? What are the time-demands which dis- 
tract from the main goals? Is there a tendency 
to short-cut significant research in favor of more 
tangible or "practical" results? Are salaries, pro- 
motions, and both the tangible and intangible 
supports of research provided according to the 
highest professional standards, and no others? 
Does legitimate professional activity need to be 
justified on the basis of nonscientific criteria? 

It is clear beyond peradventure of doubt that 
these issues need to be settled by scientists ac- 
cording to professional standards of aspiration 
and discipline which they take responsibility for 
setting: no one else is suitably qualified; no one 
else has a higher stake in the continuing exercise 
of those practices which will yield the highest 
standards of professional excellence for the or- 
ganization. These issues are similar whether the 
organization is under the aegis of a university, an 
industrial concern, or the Government. The es- 
sential value judgments in job selection are made 
on the basis of the professional identifications and 
the history of professional accomplishments 
within that organization. New organizations are 
judged on the basis of their initial program lead- 
ers, and on evidence that individual scientists can 
contribute in a responsible way toward an ideal 
destiny for an institution that has not been bound 
down by slovenly traditions, nor by the even 
heavier yoke of mediocrity. 

The ultimate level of accomplislvment and 'per- 
formance in any scientific organization depends 
not only upon the techniques evolved for helping 
it live up to the nohle ideals for which it was 
established, and the degree of aspiration and re- 



spect of its sponsors, hut it depends in large meas- 
ure upon the degree of aspiration and self-disci- 
pline exercised by its scientist members. If pro- 
fessional judgments hold sway (and this is es- 
sential), and if, in the aggregate, they are such 
as to lead to continuing internal improvement in 
the professional standards of the organization, 
that organization can withstand the impact of 
raids on its scientists, hence on its life blood, by 
competing enterprises ; it will enjoy high morale 
and both internal and external respect. Responsi- 
bility for high professional standards must be 
borne by the profession, borne with steadfast and 
depersonalized objectivity, aiming always toward 
the highest realizable levels of scientific achieve- 
ment. 

The Extension of Professional Responsibility. 
Since World War II, the impact of science (as 
realized through technology) has outstripped most 
other forces influencing business, law and politics. 
In present circumstances, the leaders of society 
and patrons of scientific enterprise are bound to be 
dependent, in all of the complex and confusing de- 
cisions relating to science, upon an adventitious 
knowledge and judgment in these affairs which is 
contributed by scientists, consultants and commit- 
tees of scientists. So much developmental action 
is possible, urgent, or even mandatory, in response 
to social, political and military needs now ex- 
pressed and for which technology can supply par- 
tial answers provided the necessary developmental 
monies can be put forward, that the combined 
Federal and non-Federal budgets are insufficient to 
support them all. 

How can these competing interests for tech- 
nology be resolved? How can resources be 
safeguarded to ensure a broad range of sci- 
entific activities, upon which the future of 
technology depends ? It is upon this jousting- 
ground of technical and scientific discrimina- 
tions that some very large social responsi- 
bilities are being transposed to scientific and 
technical consultants and committees from 
their traditional location in the hands of busi- 
ness and political leaders. This may be in- 
evitable; yet, it can be dangerous insofar as 
we may misperceive the extent of the trans- 
position of responsibility, or blind ourselves 
to the fact that wherever responsibility be 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



143 



transposed it must continue to be responsibly 
borne. Now, it is the scientist who finds him- 
self in less familiar terrain. If he acts with- 
out regard to such responsibilities, or denies 
accepting general social responsibilities for 
scientific and technical decisions he is for- 
warding, he becomes guilty of failure to as- 
sume his full and proper professional respon- 
sibility. Any denial of his bearing the full 
implications of such responsibility is as pale 
as the statement of an advertising man that he 
bears no responsibility for the tides of public 
taste nor for the creation of new marketplace 
demands. 

The displacement or transposition of re- 
sponsibility in affairs relating to science and 
technology is taking place more and more 
rapidly. There is no going backward. It 
may be that business, legal and political lead- 
ers can learn to differentiate science from 
technology, and from business enterprise, and 
to translate objectively from one code to an- 
other. To the degree that this is achieved 
such leaders deserve to continue to bear in 
full the social and political responsibilities 
and opportunities yielded by science and tech- 
nology. On the other hand, it may be that 
scientists can learn to accept and be accepted 
for a fuller share of social and political re- 
sponsibilities relating to science and tech- 
nology. This will require the scientists to bear 
a more difficult and baffling set of responsi- 
bilities than they have usually borne hereto- 
fore. To some degree both metamorphoses 
are taking place simultaneously, but as yet 
they have taken place to only a minor degree 
in comparison with the full scope of the prob- 
lem. The general pattern is one of over- 
simplification and of artful dodging of re- 
sponsibilities on both sides. A solution lies 
■first in recognizing the facts of the conver- 
gence of several different cultural patterns 
into an obligatory working relationship re- 
quired to solve important social problems, and 
second a deliberate placing of responsibility 
where it can he most fittingly borne. Any at- 
tempt to eliminate the cultural differences in 
either direction only destroys natural intra- 
cultural safeguards and interferes with the 



development of full professional integrity 
and responsibility. 
One reason why science may appear to be so 
effective in the Soviet Union is that the Russians 
consider themselves to have a scientific society. 
Therefore they encounter no conflict between 
social, political and marketplace criteria and the 
system of values and conditions essential to sci- 
ence. This tends to affect general features of their 
educational system as well as scientific and techno- 
logical enterprise. In other countries one can iden- 
tify further differences in respect to cultural inter- 
actions affecting science, as compared with either 
the Soviet system or our own. The observable 
differences are not suitable to advocate as worthy 
for use by our society, but they do reinforce the 
fact that every cultural interface pattern is an 
evolved pattern. Conscious and objective appli- 
cation to the problems we face and to the opportu- 
nities before us will undoubtedly identify a better 
destiny to set up for ourselves. Because of our 
role in the world, this discrimination and the 
social determinations it yields will be important 
for mankind as a whole. 

The Ideal of Professionalism. Our primary 
concern as scientists relates to growth in intel- 
lectual and creative power, in ourselves and our 
colleagues, to improvement in our mental grasp 
and understanding, particularly of the more gen- 
eral and comprehensive theories of science. Our 
business is thinking. Our products are con- 
ceptual, intellectual. The serious pursuit of new 
knowledge provides a special kind of discipline, 
different in important respects from that of any 
other intellectual calling. What scientists seek 
is the development of improved concepts that will 
possess pragmatic intellectual value and will stand 
testing against all valid sense experiences. The 
intellectual value concerns whether the concept 
provides a greater generalization or simplification 
of ideas, or whether it accounts more explicitly 
for the facts it relates. Although in biomedical 
science we can point to tangible evidences of 
progress in terms of new enzymes, new germs, 
and new therapeutic agents, these are by-products 
yielded through the pursuit of less tangible goals. 
A new level of understanding can be brought 
about either by beginning with an investigation 



144 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



of explicit problems (a particular disease entity, 
or a given sick person) or through the investiga- 
tion of general problems (the mode of physical- 
chemical action of enzymes, or how information 
may be genetically transmitted). In either case, 
an important clue to the scope of the ultimate 
scientific accomplishment is found in the admoni- 
tion: "There is no harm in studying a special 
subject: the harm is in doing any kind of work 
with a narrow aim and a narrow mind" (J. Hugh- 
lings Jackson, 1877) . 

There is no such thing as a logic of creativity : 
the creative process is really pre-logical. The ex- 
pository order of explanation as to what we have 
accomplished in science, and how we have tested 
our ideas, conceals the actual order of discovery. 
Logical features of science are essential for the 
logic-tight testing of new concepts against sense 
experiences. Yet the logical features of new con- 
cepts cannot be cleared up until a solution to the 
problem is evident. Not only is it necessary, in 
order to be creative, to be rid of interfering pre- 
committed opinions and prior assumptions, but 
the experience of creative work makes it increas- 
ingly difficult thereafter to hold provincial views. 
Scientific knowledge, and with it the truly pro- 
fessional scientist, can weather the shattering of 
its own fictions. Its power is such that it will 
defeat systems of thought that rest upon suasion, 
coercion or the exclusion of experience. The pur- 
suit of new knowledge has therefore a liberating 
as well as a sternly disciplining influence on the 
individual and on his community. Pessimism 
and insecurity, and a lack of confidence in being 
able to do something worthwhile in the arable 
field of science because of externally imposed 
limitations, bring a decay of intellectual and 
moral fiber. Professionalism declines along with 
the decaying of intellectual standards. 

The pursuit of professional excellence, like the 
pursuit of scientific knowledge is ennobling and 
liberating as well as disciplining. It is also ever- 
lasting. Changes in the character of the scientific 
frontier, changes in the socio-economic and socio- 
political context in which the enterprise is con- 
ducted, changes in the personnel of scientists, 
administrators and sponsors, all contribute to a 
dynamic transaction in which failure to be re- 
flective and self-critical, individually and col- 
lectively, can lead to the establishment of prac- 



tices (by direction or by default) that are likely 
to have devastating consequences for both the in- 
dividual and the organization. 

What seems to be required is a restitution of an 
ideal of professionalism as practiced, for example, 
in the medieval guilds. By this is meant an in- 
creased sense of personal dedication, a greater 
sense of individual responsibility to one's own 
work and to work of one's colleagues, an enlarged 
sense of identification through individual and 
group accomplishment, a greater thrust of pride 
in the mastery and exercise of professional skill. 
This attitude is clearly to be distinguished from 
the despairing concept of an impuissant, helpless 
cog, the contemporary dirge of one's being an "or- 
ganization man." The degree to which a sci- 
entist's professional code of action, experience 
and responsibility is disallowed obstructed or di- 
verted by his surroundings, and the degree to 
which a scientist permits this to take place, meas- 
ures certain inevitable losses of professional 
power and professional integrity. The only real 
safeguards of excellence in the conduct of re- 
search depend directly upon the professional 
characteristics of initiative, self -discipline, and 
what might be called "creative temperament.'''' 
These characteristics cannot be supplied from the 
outside. They can be, and often are, however, 
eroded and given away. It is not the hazard of 
avalanche that ruins scientific enterprise; it is the 
slow bit by bit loss of professional ideals. Ideals, 
by embodying themselves in institutions, become 
enduring historic forces. "An ideal is a picture 
of the place you will never quite, but always 
strive to reach. Its attainment happens in little 
pieces of the striving ... in any one small piece 
of honest intellectual exchange, with my neigh- 
bor, with my book ... a new beginning toward 
the unattainable is forever right at hand" (Rob- 
ert Redfield, 1955) . 



Reprise 

Biological and social evolution has gradually 
achieved greater degrees of obligate cooperation 
and interdependence within the individual, family, 
organization and society. Although each step of 
this progression has been at the expense of certain 
arbitrary modes of selfish behavior on the part of 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



145 



the individual, family, organization and society, 
this petty constriction has been counterbalanced 
by substantial gains in freedom and self-deter- 
mination at each of these levels. The progressive 
gains can be appreciated readily through an ex- 
amination of comparative physiology and be- 
havior, and through an examination of the facts 
of history over a time scale of centuries. The re- 
sultant achievements have meant substantially 
greater degrees of freedom and self-determination 
within a widening framework of cooperation, 
faith and mutual interdependence. This finds its 
expression in many phenomena : increasing urban- 
ization, increasing dependence upon federated 
activities and increasing mutual interdependence 
on the international scale. Nonetheless, develop- 
ments are not always and uniformly in the direc- 
tion of enlarging spheres of interdependence: in 
the field of international travel and exchange, 
there has been a half century of deterioration as- 
sociated with increasing formalities which are not 
entirely offset in practice by technological im- 
provements. 

It is patently true, in the aggregate, that never 
before in the history of man have there been so 
many individuals, families, organizations and so- 
cieties so inextricably interdependent. The gen- 
eralization is illustrated everywhere one looks: 
tool manufacturing, heavy industry, labor unions, 
the communications and transportations indus- 
tries, the interdependence of universities with the 
Federal Government, and many other larger and 
lesser examples. The assumption of interdepend- 
ent relations, whether contracted by individuals, 
members of a family, organizations, a given so- 
ciety, or assemblies of nations has often been 
urged and been identified on the basis of narrow 
motives and as a fulfillment of narrow and pro- 
vincial interests. The extent to which this narrow 
view obtains is regrettable; it represents an un- 
wholesome and incomplete recognition of larger 
relationships. 

The situation can be improved through the en- 
couragement and development of healthier and 
wiser motives and aims, on the part of the indi- 
vidual, family, organization, society and inter- 
national confederation. This demands the as- 
sumption of more extensive responsibilities at each 
level, the attempt to be more rational about more 
complex equations, the continuing search for and 



endeavor to achieve a higher level of integration. 
Far from this being contrary to nature, it is in 
fact an extension and more adequate representa- 
tion of vital principles that are active throughout 
all of life. These principles operate to allow in- 
creasing degrees of freedom and self-determina- 
tion. These principles are susceptible to a vast 
acceleration of their effect through conscious ac- 
ceptance of them as they apply to social existence. 
After all this is only the centennial of man's 
achieving a working conception of biological 
evolution. 

Does man have freedom to manipulate the chan- 
nels between his ideals and his actions? Can he 
choose his purposes ? Eesearch in the mental and 
neurological fields now supports the surest evi- 
dence in this regard that has even been put before 
mankind. Opportunities of consciousness and the 
degrees of freedom, of will are now known to be 
greater than we had any reason hitherto to believe. 
Freedom of choice and opportunity are ours. We 
need to utilize these not simply out of anxiety for 
individual or societal security, but for far more- 
positive reasons — reasons that take into their scope 
the whole of mankind. Freedom of the individ- 
ual, family, society and species has been increasing 
over the centuries, and now, with the advantages 
of new insights, with incentives sharpened by 
prospects of tragic and debasing alternatives, this 
freedom can be greatly extended by the indi- 
vidual, organization, society and species. This 
requires only a wider recognition and release of 
our vital biological heritage which has brought us 
to the present level of evolution and understand- 
ing. It needs only the wider recognition and re- 
lease of natural tendencies toward altruism, faith 
and mutual trust. 

To be free is to be determined by one's own 
nature. This refers equally to the individual, 
family, organization, society, or mankind as a 
whole. A wise and resolute exercise and increase 
in man's freedom through this means is the most 
direct way to liberate ourselves from the undesir- 
able exercise of our own baser tendencies. Such 
an effort can effect changes in our lives that will 
immeasurably forward us in "life, liberty, and the 
pursuit of happiness." Are we too blase to take 
seriously the high-hearted language and aspira- 
tions of our forebears? If we take such goals 



146 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



seriously, do we think that there is some easier or 
more direct way for their realization? 



Long-Kange Intramural Program 
Developments 

The Assembly or Scientists, NIMH-NINDB. 
The idea that there should be some kind of 
"faculty" organization at the National Institutes 
of Health is probably as old as the idea for estab- 
lishment of the Institutes. Such an organization 
would serve as a general forum for improving 
communication among scientists, as a means for 
formulation and expression of opinion by scien- 
tists, and as a mechanism for rendering advice and 
taking action to promote professional excellence 
and scientific achievements. As Dr. Seymour S. 
Kety pointed out, a "faculty" organization would 
provide the scientists as a whole with the same 
freedom of conscience and freedom of expression 
of opinion that has already long been afforded 
individuals. It would encourage an increase in 
the sense of participation and responsibility within 
the profession of scientists at the NIH in the 
same way that this has been cultivated by great 
universities. 

Clearly, long-range goals of the Institutes are 
inextricably bound up with the worthiness of its 
scientists ; and the professional careers of the sci- 
entists are inextricably bound up with the reputa- 
tion of the Institutes. For two years, the Labora- 
tory chiefs of the Basic Research Program, 
NIMH-NINDB, devoted their twice-monthly 
meetings to a discussion of the relative values of 
such a "faculty" organization and of ways in 
which the idea might be democratically put into 
effect. During this period, Dr. Kety provided a 
further notable contribution by recommending 
the examination of a model faculty organization, 
the highly effective Faculty Senate of the Uni- 
versity of Pennsylvania, established in 1952. 

In the spring of 1958, following thorough dis- 
cussion with the Branch chiefs of the Clinical In- 
vestigations Programs of NIMH and NINDB, 
and after discussion in executive staff meetings 
within the two Institutes, the matter was brought 
before the Scientific Directors for consideration as 
an all-NIH "faculty" organization. The Scien- 
tific Directors suggested that a trial of the idea 



should be made among the scientists of the two 
initiating Institutes ; if, after a period of experi- 
ence, the results seemed to be worthwhile, the plan 
could be considered for wider adoption within the 
NIH. The conception of an "Assembly of Scien- 
tists" was thereafter presented before an open 
meeting to which were invited all scientists of the 
two Institutes. Yet, it was another full year, 
until the Spring of 1959, before the Assembly was 
finally launched. At a second open meeting of 
scientists of the two Institutes, Officers pro-tern, 
Dr. Hal Rosvold as chairman and Dr. Karl Frank 
as secretary, were elected by secret ballot follow- 
ing open nominations from the floor. There was 
unanimous expression of interest, in principle, in 
the establishment of an Assembly of Scientists. 
The chairman pro-tem was instructed to appoint 
a committee to draft a constitution which would 
then be presented to the scientists. In due course a 
Provisional Constitution was adopted, and in the 
fall of 1959 a revised constitution was formally 
adopted by a mail vote of 230 members of the sci- 
entist staff of the two Institutes. They also ex- 
pressed their wish voluntarily to participate in 
future activities of the Assembly. 

As far as is known, such a "faculty" organiza- 
tion within the Government is without precedent ; 
yet this is not surprising in view of the fact that 
the National Institutes of Health are themselves 
without parallel in mission or spirit of organiza- 
tion within the Government. The potential value 
of this Assembly to the ultimate stature of the 
Institutes, and to the level of professional regard 
in which the scientists themselves will be held, is 
limited only by the vision, determination, and 
willingness of members of the Assembly to assume 
individual and collective responsibility of a con- 
structive nature. Through the Assembly they 
have a unique opportunity to create an example 
for scientists elsewhere at the NIH and more gen- 
erally throughout the Federal Government. 

The Principle of "Tenure." Although employ- 
ment security in the Government is accorded em- 
ployees in all categories after only one year of 
employment, one year is too brief a period in 
which adequately to develop or evaluate the skills 
of junior scientists. If tenure were to follow 
automatically whenever a scientist works for a full 
year at NIH, three disagreeable alternatives would 
be forced: (1) either the Institutes would have to 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



147 



be expanded indefinitely, or (2) there would be 
inadequate space for essential research operations 
after only 2 or 3 years of such practice, or (3) 
there would be no opportunity to provide research 
training for aspirant scientists. Since the most 
effective scientists are often asked to fill attractive 
research and teaching posts elsewhere, there is a 
continuing risk of losing the best research talent 
from the Institutes, the very leaders with whom 
aspirant scientists seek to study, the ones who 
chiefly account for the professional reputation of 
the Institutes. It is therefore imperative for saf e- 
guarding the professional stature of the Institutes, 
the opportunity for scientists to pursue research 
effectively, and for junior scientists to receive an 
adequate foundation in research training, that all 
of the scientists participate in a definite plan 
whereby the younger scientists can be provided 
training and experience in research for definite 
but limited periods of time, e.g. for from 2 to 3 
years. Although this period might be extended 
for an additional year in exceptional cases, that 
would ordinarily be the final limit. The only 
possible exceptions would be vacancies resulting 
from the retirement or departure of senior scien- 
tists. "Tenure" is therefore to be effected by estab- 
lishing a firm understanding, prior to the employ- 
ment of a new junior scientist, of the time-limited 
nature of his appointment. The practice of a 
policy of "tenure" is so standard throughout the 
academic world that it is widely understood and 
respected by scientists. Such pre- vision precludes 
the embarrassment and misunderstanding which 
otherwise may arise among scientists, each of 
whom is devoting his utmost energies to research, 
and for whom, inevitably, there can be only 
limited local research resources. 

The Principle or "Sabbatical" Leave. Creative 
scientific endeavor demands mastery of subject 
matter and exercise of initiative, self-discipline, 
and personal devotion at a level that cannot be 
sustained indefinitely without intellectual refresh- 
ment and revivification. Anyone attempting 
highly creative scientific work, with the intense 
preoccupation and internal involvement that this 
entails, tends to "go stale" without periodic relief 
in the form of opportunities to renew his mas- 
tery of the field, to learn new technical and con- 
ceptual skills, and to obtain a new perspective on 



scientific values relating to his work. To some 
degree this kind of "change in pace" is effected by 
the individual investigator within his normal 
working pattern; nonetheless, over a span of 
years, he is likely to become even less aware of the 
conceptual strictures which may impoverish his 
accomplishing more effective and creative endeav- 
ors. Universities of high standing have long 
recognized as prominent among the essential re- 
quirements for sustained high-quality creative 
scholarship the need for senior faculty members to 
be given extended periods of time away from regu- 
lar duties, usually at 7-year intervals. 

The Laboratory chiefs of the Basic Research 
Program initiated discussions on this subject and 
were encouraged by the Director of NIMH, Dr. 
Robert H. Felix, to draft plans for a "Sabbatical" 
Leave Program. Under the chairmanship of Dr. 
David Shakow, an NIMH committee established 
the essential administrative considerations for 
such a program which was then endorsed, in 
principle, by Dr. Felix, Dr. Pearce Bailey, Direc- 
tor of NINDB, and by the Scientific Directors. 
Authority for this practice is found in existing 
regulations providing for the Work Assignment 
and for the Training of Institute scientists. The 
"Sabbatical" Leave Program will provide the sen- 
ior scientists belonging to the permanent staff, 
upon whom the Institutes stake their research mis- 
sion and reputation, an opportunity at 7-year in- 
tervals to engage in sabbatical activity of their 
own choice. Dr. Harris Isbell was the first scien- 
tist to be sent on this new leave program; others 
in the two Institutes are already on leave or are 
proceding with plans for participation in this 
augmented opportunity for personal intellectual 
growth and career development in preparation for 
further creative work at the National Institutes of 
Health. 

Educational Programs Relating to the NIH. 
"Scratch a scientist and you will find a teacher." 
An important aspect of professional activity is 
helping others to acquire intellectual and tech- 
nical skills and to have experience in the extension 
of these skills to new frontiers. The most charac- 
teristic form of such "profession" takes place be- 
tween scientist-preceptors and their junior col- 
leagues. Nearly every collaborative research 
undertaking involves similar vital intellectual 



556044—60- 



-11 



148 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



exchange, a function intrinsic to the life of an 
investigator. 

Early in the history of the National Institutes 
of Health there was an expression of need for 
more formal and organized opportunities for par- 
ticipation in both directions in the educational 
process. The NIH established a branch of the 
U.S. Department of Agriculture Graduate School 
which has grown steadily in attendance and 
autonomy. The Research Associates Program, 
established 3 years ago, has added to the preceptor- 
apprentice relationship a complementary means 
for a broad-based education in bio-medical re- 
search, through the provision of course work and 
seminars extending into fields other than the As- 
sociate's primary specialization. This last year, 
members of the NIH Scientific Advisory Com- 
mittee established a non-profit corporation, The 
Foundation for Advancement of Education in the 
Sciences, Inc., which will facilitate a further ex- 
tension of educational opportunities at the NIH. 
Dr. Daniel Steinberg is chairman of the Board of 
Directors of the new Foundation. The Board also 
includes other representative leaders in the field 
of science education outside the NIH. The 
Foundation, like the Graduate School Branch it 
takes over, will be largely self-sustaining from tui- 
tion. The increase of intellectual experience which 
the Foundation can provide will undoubtedly 
prove beneficial to the recruitment and sustained 
intellectual vigor of scientists who derive insight 
and satisfaction from participating in professional 
educational activities. 

The Construction of a Greenhouse Facility. 
For several years scientists in the Laboratory of 
Cellular Pharmacology have expressed their need 
for a facility for direct investigation of alkaloid 
synthesis in plants. During the year a neat little 
greenhouse was constructed and put into opera- 
tion under the direction of Dr. Harvey Mudd. 
Several alkaloids, particularly those related to 
groups of tranquillizers, psychotomimetic drugs, 
narcotic agents, depressives, and cerebral stimu- 
lants can now be studied in relation to their me- 
tabolic precursors and the ways in which they are 
handled and inactivated by plants. It will also 
be possible, by this means, to label complex com- 
pounds by feeding plants with radioactive build- 



ing blocks, in many cases saving difficult labora- 
tory synthetic procedures. 

The first procedures undertaken by Dr. Mudd 
and his colleagues have established certain com- 
mon features of metabolic pathways which are 
common to higher mammals, single celled organ- 
isms, and higher plants. This revelation confirms 
that it will be practicable to examine a number of 
complex metabolic pathways first in plants where 
the growth and harvesting of large quantities of 
particular metabolic steps will facilitate solution 
of a number of important problems. With cer- 
tain key steps established in plants and with 
knowledge of the essential substrates, enzymes, co- 
factors, etc., it will be possible to confirm and ex- 
tend these findings much more quickly in higher 
mammals. Members of the laboratory will now 
be able to work back and forth between plant and 
animal biochemistry and to look for variants and 
consistencies over a very broad biological field of 
endeavor. 



General Commentary 

This year has witnessed a continuing harvest of 
outstanding research papers from the Basic Re- 
search Program. The entire enterprise can 
be readily justified on the basis of a few of the 
really creative ideas brought forth. The status 
of the program is also measured by the large num- 
ber of invitations which come to its scientists to 
provide papers for national and international 
meetings, and to lecture before or to join the facul- 
ties of outstanding universities. Judging by the 
increasing qualifications of scientists seeking posi- 
tions here, it is evident that the program is gaining 
in reputation as an intellectual and experimental 
resource for effective scientific training and experi- 
ence at all levels. Nearly every major university 
in this country and some seventeen foreign uni- 
versities are represented by one or more scientists 
employed in the program this year. 

Dr. Mortimer Mishkin was sent to work for 3 
months at the Nencki Institute in Warsaw, 
Poland, and Dr. Stefan Brutkowski of that In- 
stitute has been sent by the Polish Academy of 
Science to work with Dr. Mishkin and Dr. Hal 
Rosvold for about a year as a guest worker. 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



149 



Altogether, 16 of our scientists were sent abroad 
for periods of work and intellectual exchange 
during the year. There were nine scientists in 
the program who attended international meetings 
outside this country. Distinguished scientists 
from more than 20 different countries visited the 
Basic Research Program this year. 

One of the traditional ways of improving the 
creative power of an organization — through the 
use of expert consultants — has been actively ex- 
ploited this year as in the past. The 12 members 
of the Boards of Scientific Counselors of the 2 
Institutes, NIMH and NINDB, have continued 
to give encouragement, intellectual stimulation, 
and to provide programmatic as well as scientific 
advice. Some 37 other expert consultants parti- 
cipated and advised in relation to special aspects 
of the program. Professor Torsten Teorell of the 
University of Uppsala, Sweden, and Professor 
Ulrich Franck of the Max Planck Institute in 
Darmstadt, West Germany, came to work for a 
period of time with Drs. Ichiji Tasaki and Con- 
stantino Spyropoulos at the Woods Hole Marine 
Biological Laboratory this summer. Similarly, 
Drs. Sydney Brenner and Francis H. C. Crick 
from Cambridge, England, paid working visits 
to Dr. Bernhard's Section on Physical Chemistry. 
Altogether, about a dozen foreign scientists spent 
working periods in the program. Through a 
chance encounter with Dr. Emanuel Piore, Di- 
rector of Research for the International Business 
Machines Corporation, an exceptionally exciting 
research collaboration has been arranged. Dr. 
Sidney Bernhard introduced a group of engineers 
and mathematicians of the IBM Research Divi- 
sion, visiting Bethesda at Dr. Piore's suggestion, 
to the conceptual problem of "breaking the code" 
for the nucleic-acid sequencing of amino acids in 
genetic transmission, and more generally in all 
protein synthesis. Dr. Bernhard and Dr. Dan 
Bradley had conceived of a way in which the bulk 
of the presently tedious chemical identifications 
of one after another of the amino acids in serial 
order could be short-cut by utilizing advanced 
electronic computers in elaborate logical analyses. 
Dr. William Duda of IBM has since then been 
devoting full time to the difficult mathematical 
end of this investigation. Hs has been able to put 



to work the best IBM computer programmers and 
to commit the most modern computer equipment 
for this purpose. In September Dr. Duda ac- 
companied Dr. Bernhard to Copenhagen to par- 
ticipate in discussions on this subject at the 
International Symposium on the Genetic Control 
of Protein Synthesis. Dr. Bernhard had been 
invited by the Symposium Program Committee 
to give an address at the Symposium in replace- 
ment of Professor Linderstr5m-Lang, an inter- 
nationally famous Danish scholar noted for his 
work in this field, who died unexpectedly last 
spring. 

The project to "break the code" of amino-acid 
sequencing by this means is still in early stages 
of development. Each step thus far has pro- 
ceeded favorably, but it is a "long shot" as to 
whether the concepts and techniques may prove 
successful : nonetheless, both IBM and the Basic 
Research Program are confident that whatever is 
learned along this important line of investigation 
will be worthwhile. Whereas it now takes years 
of conspicuously conscientious and compulsive 
chemical work to determine the sequence of amino 
acids in even relatively small proteins, the new 
method shows promise of reducing this time to a 
matter of a few weeks. If this turns out fa- 
vorably it will vastly accelerate the analysis of 
differences in the almost countless proteins of im- 
portance to biology and medicine, and it will 
make practicable the identification of the sites of 
defect of genetically determined developmental 
and metabolic errors. 

During the year Professor Leo Szilard pub- 
lished the theory of aging which he developed 
while serving as a consultant to the program. He 
now has in preparation two new and equally chal- 
lenging papers on the theory of antibody forma- 
tion. After long deliberation, Dr. Szilard 
declined employment in this program in favor of 
accepting a long-term NIH extramural grant 
which would allow him to retain his post at the 
Enrico Fermi Institute in the University of Chi- 
cago and still to collaborate with our staff for ex- 
tended periods as a guest worker. Unfortunately, 
the same week that Dr. Szilard was informed of 
favorable action on the NIH grant, he also 
learned that he has a highly malignant tumor 
which is effectively inoperable. 



150 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



A Personal Note 

Prior to appointment as Director of Basic Re- 
search for the two Institutes, I discussed with 
everyone concerned a deliberate limitation on the 
length of time I felt it was reasonable to commit to 
such heavy administrative responsibilities. This 
year, shortly before the completion of 3 years in 
office, I asked permission of the two Institute Di- 
rectors to be relieved from this work "in the near 
future." My reasons for adhering to such a time 
limit are threefold : First, I believe it desirable for 
a scientific program to have a change in leader- 
ship from time to time. Dr. Kety has already set a 
precedent for this. Any leader of a research group 
has conceptual limitations which are likely to be- 
come an increasing interference to the program as 
his time in office is stretched out beyond about 3 to 
5 years. Second, as Ian Stevenson puts it, "The 
possession of the power to make decisions can 
eventually persuade anyone that he also has the 
proper knowledge to do so." My third reason for 
wishing to adhere to such a time limit derives from 
a very personal desire to continue full-time re- 
search. 



BASIC RESEARCH— (NIMH-NINDB) 



Laboratory of Biophysics 

The central objective of the Laboratory of Bio- 
physics is to understand the nature and the im- 
plications of the ion movements fundamental to 
the initiation and propagation of a nerve impulse. 
The staff— John W. Moore, Richard Fitz-Hugh, 
Robert E. Taylor, William J. Adelman, W. Knox 
Chandler, John H. Gebhart, and Ernest R. Whit- 
comb — have continued to progress towards this 
objective, in part with the collaboration of the 
Naval Medical Research Institute, and the Com- 
putation Laboratory, National Bureau of 
Standards. 

The characteristics of individual ionic move- 
ments as first determined from measurements of 
the squid-axon-membrane current and potential 
under controlled electrical, geometrical, and ionic 
conditions have led to far-reaching conclusions. 
But subsequent work has made it necessary to 



undertake an examination of the extent to which 
the measured membrane currents depend upon 
the adequacy of these controls. The records and 
analyses generally confirm the 1958 preliminary 
conclusions that the membrane potential of strong 
axons could and had usually been reasonably well- 
controlled and that the qualitative characteristics 
of these axons were similar to those of less-diffi- 
cult axons. The conditions for an adequate con- 
trol have been found to be more stringent and 
the understanding of failures to be more difficult 
than had been anticipated. The confusing effects 
of instability can be reduced by restricting the 
membrane current measurements to the region of 
best potential control but an estimate for the accu- 
racy of control must await further investigations 
of the resistance between the control electrodes 
and the capacity of the membrane. 

The sodium potential of Hodgkin and Huxley 
measured by voltage clamp may be assumed to be 
as good a measure for sodium-ion concentration 
changes inside the axon as it has been found to be 
for those outside. Thus measured, the net sodi- 
um inflow of the surviving axon at rest and the 
increase by stimulation are in general agreement 
with isotope measurements. The net flow of sodi- 
um is reversed by voltage pulses above the sodium 
potential as predicted by the sodium theory. By 
the same procedure, it was found that ten precent 
of normal external divalent ion concentration in- 
creased the resting net sodium inflow to about 
three times the normal value. 

The phenomena of finite and infinite trains of 
impulses, anodal break excitation, refractoriness, 
and accommodation depend upon certain mathe- 
matical properties shared by a large class of sys- 
tems, electronic and chemical, as well as 
physiological. The well-known Van der Pol relax- 
ation oscillator equations have been generalized to 
include these phenomena and the Hodgkin-Huxley 
squid-axon equations have been reduced to a quali- 
tatively similar form. The generalized equations 
can be represented on a plane with regions cor- 
responding to the physiological states described 
by classical neurophysiology. Thus, a basis for 
deeper and more useful understanding of well- 
known phenomena is emerging. 

Digital computations for a medullated axon 
with a modified Hodgkin-Huxley membrane at the 
nodes give an impulse velocity of 11.9 m/sec for 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



151 



various super threshold stimuli but the long 
latency at threshold requires much more expensive 
computer runs than have yet been undertaken. 
Analog computations have shown that with rea- 
sonable modifications, the Hodgkin-Huxley squid- 
axon equations produce action potentials agreeing 
closely with those obtained by Dr. John Dalton 
from a lobster axon in normal and excess potas- 
sium media. 

In a joint project, with Dr. Seymour L. Friess, 
NMRI, on the action of synthetic acetylcholine- 
sterase inhibitors, pure optical and geometrical 
isomers in the ethylenediamine series and in the 
1,2-aminocyclohexane family have been compared 
by in vitro enzyme inhibition and nerve blockage. 
Although both lines of investigation are interest- 
ing and promising, the results are so increasingly 
divergent as to further decrease the probability of 
a casual relationship between the two processes. 
A shift of emphasis from desheathed whole nerve 
to single node preparations is giving the results a 
clarity not possible when the drugs diffuse to many 
sites of action. 

Laboratory of Neuroanatomical Sciences 

The Laboratory of Neuroanatomical Sciences is 
organized into five sections, one a field station in 
Puerto Rico. Each section chief has composed a 
summary of the section's research activities during 
1959. The research of each section is sufficiently 
unique to make an overall summary of activities 
less meaningful than presentation of separate 
accounts. 

SECTION ON DEVELOPMENT AND 
REGENERATION 

Research activities of the Section on Develop- 
ment and Regeneration fall into categories of (a) 
neurogenesis, (b) regenerative potentialities of 
central and peripheral neurons, and (c) experi- 
mentally induced structural alterations in the cen- 
tral nervous system. 

(a) Drs. Sidman, Miale, and Feder incorpo- 
rated tritium-labeled thymidine into deoxyribonu- 
cleic acid (DNA) of cells preparing for division. 
Autoradiography with tritium-labeled thymidine 
(thymidine-H 3 ) provided a method of marking 
such cells in the relatively inaccessible mammalian 
embryo. Pregnant mice were injected intrave- 



nously with thymidine-H 3 and killed at various 
intervals. Autoradiograms were prepared of sec- 
tions through the embryonic brains. Eleven-day 
embryos, fixed 1 hour after exposure to thymi- 
dine-H 3 , showed heavy labeling of most cell nuclei 
in the external half of the primitive ependymal 
layer in the wall of the cerebral vesicle, and almost 
no labeling in the inner half. Thus the external 
half of the primitive ependymal layer was a site of 
DNA synthesis. Six hours after exposure to thy- 
midine-H 3 , the labeled nuclei occupied the inner 
(ventricular) half of the primitive ependymal 
layer, and most mitotic figures at the ventricular 
surface contained labeled chromosomes. Forty- 
eight hours after injection, labeled nuclei had mi- 
grated laterally ; some had entered the developing 
mantle layer, but many remained in the primitive 
ependyma and had repeated the cycle of DNA 
synthesis, migration, and division. Development 
of the primitive ependyma was similar through- 
out the embryonic nervous system. The cells of 
the primitive ependymal layer behaved synchro- 
nously. The primitive ependymal layer is a pseu- 
dostratified columnar epithelium within which 
nuclei of undifferentiated cells migrate to and fro 
in relation to the mitotic cycle. 

It was found that the primordium of the cere- 
bellum made its appearance after 11 days of ges- 
tation. At this time 3 zones comprising the pri- 
mordium were recognizable. Cells of the epen- 
dymal region migrated into the intermediate zone 
to participate in formation of intracerebellar nu- 
clei. Development of the various components of 
the cerebellum of mice was traced on different 
days of gestation. The cells destined to become 
Purkinje neurons migrated from the primitive 
ependymal region to their definitive positions in 
the future cerebellar cortex and rapidly differenti- 
ated there. 

(b) Drs. L. Guth and C. J. Bailey evaluated the 
relative roles of the sympathetic and parasympa- 
thetic pupillary nerve fibers to determine whether 
autonomic neurons can maintain the function of 
autonomic effector organs other than those that 
they normally innervate. The atropinized pupil 
dilated significantly in darkness and a significant 
portion of this dilation was abolished by sym- 
pathectomy. Contrary to current opinion the sym- 
pathetic nervous system plays an active role, rather 
than merely a tonic one, in maintaining pupillary 



152 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



dilation in darkness. They found that pupillary- 
size was partially restored under the influence of 
"foreign" nerve roots, although these roots did not 
mediate a "darkness reflex." This was accom- 
plished by transecting the sympathetic rami of T 1 
to T 3 to allow collateral sprouts from rami of T 4 
to T 7 to innervate the pupillary ganglion cells of 
the superior cervical sympathetic ganglion in cats. 
The nerve fibers apparently retained their original 
specificity, inasmuch as the heterogenously rein- 
nervated pupil dilated in response to decreased 
environmental temperatures. 

Dr. Guth, with Drs. James Campbell of Colum- 
bia University and Lamar Soutter of Boston Uni- 
versity, are testing in monkeys whether or not dia- 
phragmatic function can be maintained by the re- 
current laryngeal nerve. The proximal recurrent 
laryngeal and distal phrenic nerve segments were 
anastomosed. Regeneration has been in progress 
for nearly 1 year. Dr. Karl Frank of the Lab- 
oratory of Neurophysiology will cooperate with 
Dr. Guth early in November to determine electro- 
physiologically the state of the diaphragmatic re- 
innervation in these animals. Dr. Guth previously 
conducted a similar study in rats and demonstrated 
that vagophrenic anastomosis did, indeed, restore 
function to the denervated hemidiaphragm. If 
the experiment in the monkey succeeds, it will 
be possible to consider application of the operative 
procedure to those human disorders characterized 
by pathology of the phrenic nerve. 

The chief of the section and Dr. E. R. Feringa, 
collaborating with Drs. J. B. Campbell, A Bassett, 
and C. Thuline of Columbia University, have con- 
tinued to study regeneration in the mammalian 
spinal cord. A study of transected spinal cord of 
16 monkeys with gaps of several millimeters was 
carried out. In a report of this work presented at 
the April meeting of the American Academy of 
Neurology, postulates for evaluating regeneration 
in the central nervous system were laid down. 
These are: There must be proof that the spinal 
cord was severed; clinically observable signs of 
restitution of motor and/or sensory functions; 
proof of physiological conduction across the healed 
lesions, including electrical recordings from the 
spinal cord following stimulation above and/or 
below; trans-synaptic potentials must be demon- 
strated ; finally, there must be unquestionable veri- 
fication of reestablishment of histological continu- 



ity. All of these are needed in one and the same 
animal. To date, no single experiment in this or 
any other laboratory has fulfilled all these postu- 
lates in mammals. Current studies in cats and 
monkeys are in progress combining the techniques 
of surrounding the lesion with Millipore to pre- 
vent encroachment of connective tissue, and treat- 
ment of the animals with Piromen. Multiple 
short segments of frozen-dry, peripheral-nerve 
homografts, have been implanted in some. A pre- 
liminary operation to fuse the skeletal elements of 
the spine prior to spinal-cord transection has led 
to improved histological and physiological results 
in cats. By combining several procedures, each of 
which has some demonstrated merit, it is hoped to 
achieve fulfillment of our postulates. 

Drs. Feringa, Campbell, Bassett, and Thuline 
have explored methods of grafting segments of 
large peripheral nerves, using the goat as the 
principal laboratory animal. Homologous nerve 
segments, banked by freezing, freezing and ir- 
radiation, and freezing and drying, were im- 
planted in gaps produced surgically in the sciatic 
nerve of these animals. Preliminary studies in 
cats, with smaller nerves, demonstrated swift func- 
tional and anatomical regeneration across gaps of 
1 cm., bridged with frozen-dry segments of nerve 
wrapped in Millipore sheaths. It is hoped that 
the studies in goats will provide information ade- 
quate to permit the method to be extended to 
human subjects for the repair of large peripheral 
nerve gaps. 

(c) Studies on alteration of the structure of the 
central nervous system resulting from asphyxia 
neonatorum and nitrogen asphyxiation of newborn 
and young monkeys have been carried out in col- 
laboration with members of the field station, Sec- 
tion on Perinatal Physiology, in San Juan, Puerto 
Rico. 

Asphyxia neonatorum was induced in monkeys 
near term by detaching the placenta at hystero- 
tomy under local anesthesia, keeping the fetal 
membranes intact. Eleven to sixteen minutes 
later the fetuses were delivered from their mem- 
branes and resuscitated by pulmonary insufflation 
with oxygen. The infant monkeys showed neuro- 
logical deficits during life. Five were killed by 
perfusion-fixation at 2 to 9 days of age. Brains 
of these and of two which were not asphyxiated 
were studied. A common pattern of structural al- 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



153 



teration was encountered in the nervous system of 
the asphyxiated monkeys. Nuclei were symmetri- 
cally affected ; those most consistently and severely 
damaged were the nucleus of the inferior col- 
liculus, gracile and medial cuneate nuclei, roof 
nuclei of cerebellum, ventral posterior group of 
thalamic nuclei, globus pallidus, putamen, and 
vestibular nuclei. The cerebral cortex was 
severely damaged in only one monkey. Lesions 
began with primary nerve cell and, less frequently, 
neuroglia cell lysis and loss. Secondary damage 
of myelin sheaths, and reactions of astrocytes, 
endothelial cells, vascular adventitial cells, and 
phagocytes were noted. A relation of lesions to 
vascular distribution was not apparent. Hemor- 
rhages were not encountered. 

Six newborn monkeys and four juveniles were 
asphyxiated in N 2 , resuscitated similarly (cardiac 
arrest in one) , and killed 10 days later. Appro- 
priate controls were provided. Nitrogen asphyxi- 
ation failed to produce the pattern of brain dam- 
age seen after fetal asphyxia. No focal changes 
were observed 10 days after cardiac arrest. As- 
phyxia neonatorum and N 2 asphyxiation have 
quite different effects on the central nervous sys- 
tem of Macaca mulatto,. 

Guinea pig fetuses were asphyxiated at term by 
clamping the uterine blood vessels during lapa- 
rotomy and resuscitated by insufflation of their 
lungs. Observations of degree of asphyxia and 
degree of resulting neurological deficit were made. 
Asphyxia produced neurological deficits at least 
transiently. Asphyxiated animals and their con- 
trol litter-mates were tested in an alternation maze 
during the first week of life or during the 12th 
week. Some of these were tested also in a water 
maze at about 18 months of age. Asphyxiated 
animals made more errors than the controls in 
learning, although relearning tests did not show 
that they forgot more rapidly than controls. 
There was no correlation of learning performance 
and degree of asphyxia. Histological examina- 
tion of the brains revealed damage similar to, 
though less severe than, that reported previously 
(1942). Hemorrhages were seen only in acute 
stages. Chromatolytic changes in neurons, loss of 
neurons, and neuroglial reactions appeared in the 
brain stem and thalamus. Circumscribed bi- 
laterally symmetrical loci of damage were en- 
countered in the thalami of long-term animals. 



The signs of damage were less marked in animals 
living for a year or more than in those dying dur- 
ing the first few days or weeks. The neuroglia 
cell changes subsided with time. Long-term sur- 
vival animals exhibited minimal brain damage. 
This experiment yielded further evidence that 
asphyxial episodes at birth produce neurological 
deficits, learning defects, and brain damage. It 
casts doubt on the thesis that the short asphyxial 
episodes seen clinically do not cause some undesir- 
able sequelae. 

SECTION ON NEUROCYTOLOGY 

As the Section has developed, its interests and 
problems investigated fall into two categories: 
morphological and chemical. 

In the first group our investigations this year 
have been directed toward learning more about 
the relationship between the neuron and its sup- 
porting structures, the glia in the central nervous 
system and the capsular cells in the sensory 
ganglia. 

In the peripheral ganglia each neuron is en- 
closed within a capsule consisting of Schwann 
cells. In the dorsal root ganglia, the capsule 
characteristically is closely apposed to the surface 
of the neuron except in certain spots where the 
two surfaces diverge, forming bays or lakes into 
which the capsular cell sends protruding ridges 
and microvilli. These spots are associated with 
intracellular vesicles in the capsule. It is possible 
that these formations are related to ionic move- 
ments during the activation of the cell surface 
which is part of impulse propagation. In the 
ganglia of the eighth nerve, myelin coats many of 
the neurons. We have studied these in the gold- 
fish. The myelin occurs with various degrees of 
compactness. Nodes of Ranvier are absent. The 
presence of myelin completely surrounding a neu- 
ron raises the question of transport of nutrients 
and exchange of metabolites between the neuron 
and the extracellular space. Such cells are per- 
haps more amenable to physiologic studies than 
those of the central nervous system where extracel- 
lular space is also limited by the close packing of 
all elements. 

Dr. Brightman and Dr. Albers have completed 
a histochemical study of species differences in the 
distribution of cholinesterase activity in the 



154 



ANNUAL REVIEW OP INTRAMURAL RESEARCH 



central nervous system. Pseudocholinesterase oc- 
curs in the vascular endothelium in the rat, gold- 
fish, and toad, but in the glia of the cat and rooster. 
The significance of this distribution is not clear 
at present. 

The chemical studies of this section have been 
carried out under the direction of Dr. Albers. 
His investigations have been concerned with the 
role of y-aminobutyric acid in the metabolic reac- 
tions of the brain. This study is based upon the 
observation that y-aminobutyric acid and its di- 
rect precursor, glutamic acid, occur in large 
quantities in the brain. The enzyme which 
catalyzes the conversion of glutamate into GABA 
is localized in gray matter, possibly in the 
neurons, and is not found in other tissues of the 
body. 

The section has been host this year to Dr. John 
Hills, NINDB trainee, and Dr. Mary Grillo, re- 
search fellow, both of whom will be studying cer- 
tain aspects of the peripheral nervous system. 
Dr. L. Embree has joined with Dr. Albers as a 
Research Associate. 

Dr. Palay gave a Phillips lecture at Haverford 
College in Haverford, Pennsylvania, and also 
lectured at the Rockefeller Institute, New York; 
the University of Washington, Seattle; Columbia 
University, New York; George Washington 
University Medical School, Washington, D.C. ; 
Annual Symposium of the Gastroenterological 
Research Group, Atlantic City; and in the Lin- 
nean Society, London. Dr. Albers participated in 
a national conference on "Inhibition in the Cen- 
tral Nervous System and y-aminobutyric acid." 

SECTION ON EXPERIMENTAL 
NEUROPATHOLOGY 

Several series of studies were completed as part 
of a long-range plan to establish extra- and intra- 
spinal factors concerned with maintaining normal 
spinal-cord structure and function. 

A comparative miscroscopical study of the 
vascular system revealed the widespread distribu- 
tion of intervascular connective-tissue fibers in 13 
animal species, including monkey, dog, raccoon, 
cat, rabbit, several rodents, opossum, and pigeon. 
The significance of establishing such a fibrillary 
system in animals is (a) normally vessels tend 
to undergo involuntary changes which should not 



be interpreted as due to experimentation, dis- 
ease, or aging, (b) such a fibrillary system con- 
tributes to the stability of normal vasculature, 
(c) such intervascular connections may have a 
deleterious effect on blood circulation in cerebral 
edema when blood vessels are stretched and their 
lumens distorted, and (d) differences in regional 
susceptibility may be ascribed to variations in 
number and size. 

Another study, aimed at the oligodendrocytes 
along the vessels, disclosed that in all animals 
these cells are arranged in rows, small groups, or 
larger clusters near perivascularly arranged neu- 
rons or near points of arborization of vessels. 
None is arranged in a manner to support older 
concepts that these cells are concerned with the 
function of neuronal perikarya or processes. On 
the other hand the particular arrangement of 
these cells suggested that they are concerned with 
intrinsic control of blood flow to neurons. The 
complex arrangements of oligodendrocytes and 
vessels were more striking in the spinal cord ; this 
observation may point to the need for a more in- 
tricate control mechanism in this organ than the 
brain to permit utmost economical distribution of 
blood between activated neurons. These results, 
presented at the Conference on Microcirculation 
in Physiology and Pathology May 4, 1959, at the 
NIH, have opened new avenues for anatomical, 
histochemical, physiological, and pathological in- 
vestigations. They have initiated further re- 
search both to determine the exact distribution in 
a tridimensional model of the central nervous 
tissue and to assess the reaction of these cells 
under abnormal conditions. 

As part of an investigation on the response of 
the tissue to abnormal capillary blood flow, a dog 
material injected with oil was studied and report- 
ed jointly with Dr. Roy L. Swank, University of 
Oregon, Medical School, Portland, Oreg. The 
tissue changes, which varied greatly in size, were 
always the result of capillary obstruction. The 
tendency of perivascular conversion of fibrinogen 
to fibrin was less striking in dog than in man. 
Two other observations were : a striking diffusion 
of iron as indicative of disturbed iron metabolism 
and an aggregation of oligodendrocyte nuclei in 
small foci of necrosis where neurons had disap- 
peared indicating different resistance of oligoden- 
drocytes and neurons to ischemia. 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



155 



The significance of abnormal staining of neurons 
was reviewed. An extensive study of histological 
material and of previous publications formed the 
basis of a brief report given before the American 
Association of Neuropathologists (Annual Meet- 
ing, Atlantic City, June 1959) and of a larger 
review to be published in Ergebnisse der Anato- 
mie. Conclusive evidence of the artifactual na- 
ture of dark neurons was given. As a conse- 
quence, contemporary views on the role of 
so-called dark neurons during normal activity 
and disease reaction could not be confirmed. 
Only material free of dark neurons should be used 
for cytological and neuropathological studies. It 
was gratifying that principles of fixation and 
autopsy were established which prevented normal 
neurons from undergoing artifactual changes. 

In order to understand how to control the fac- 
tors involved in histological preparation a series 
of measurements was performed. With the aid 
of karyometric methods it was possible to demon- 
strate that the size of neuroglial nuclei was greatly 
influenced by fixatives. The shape and staining 
of nuclei, in particular of astrocyte nuclei, varied 
with the technique used. With some of the pro- 
cedures the astrocyte nuclei were rounded where- 
as with the best procedure available they exhibi- 
ted an extreme degree of pleomorphism. This 
pleomorphism occurred in most regions and ani- 
mal species except several of the small rodents. 
For investigations concerned with identification 
of astrocyte and oligodendrocyte nuclei only such 
species should be selected where astrocyte nuclei 
are easily identifiable because of their pleomorphic 
shape, as in the chinchilla. 

As a consequence of these studies all histological 
material is now prepared according to a standard 
procedure: (a) use of a modified Heidenhain's 
Susa solution as perfusate for fixation; (b) delay 
the autopsy 4 hours after the perfusion; (c) im- 
mediate transfer of blocks to alcohol; and (d) 
staining of the microscopical sections by the peri- 
odic acid-Schiff procedure and then immersion in 
gallocyanin-chrom alum, pH. 1.7, at room tem- 
perature for 24 hours. 

Two series of animals were studied by Dr. 
Helen D. Ramsey to establish the distribution of 
extradural fat ; one concerned the conditions in the 
cat from newborn to adult and the other a com- 
parative study in the rabbit and monkey. This 



is the first systematic study of the intricate manner 
in which fat is deposited in several animal species. 
The extradural fat permits the complex move- 
ments of the vertebral column without tearing 
the spinal cord and rootlets. 

The Section has been host to Dr. Mignon Malm 
(University of Stockholm, Sweden) during 1959. 
Dr. Helen D. Eamsey left the Section during the 
current year. 

SECTION ON FUNCTIONAL 
NEUROANATOMY 

Professional personnel of the Section of Func- 
tional Neuroanatomy are: Richard Gacek and 
Grant L. Rasmussen, Chief. This section con- 
cerns itself primarily with nervous pathways and 
connections of the brain and spinal cord. 

In order to better understand the neural mech- 
anism of hearing, studies of the auditory afferent 
system, so long neglected, have received particular 
attention. Point-to-point interneuronal relation- 
ships existing between the organ of Corti and the 
cochlear nucleus, and the manner of projection 
from the latter to higher auditory nuclear groups, 
have been restudied in more detail than hereto- 
fore by the experimental anatomical approach. 
The study dealing with the projection of cochlear 
nerve fibers on the cochlear nucleus has been com- 
pleted for both the cat and chinchilla. The special 
arrangement of the nerve terminals and mamier of 
distribution to the different subnuclear groups of 
the cochlear nucleus have been determined and 
correlated with the sites of lesions. This informa- 
tion has been brought together in the form of a 
plastic model of the cochlear nucleus which per- 
mits one to gain a three-dimensional view of the 
course and distribution of cochlear nerve fibers 
that transmit nerve impulses from the different 
tonotopic regions of the organ of Corti. 

The efferent or recurrent connections of the 
cochlear nucleus have been studied extensively in 
the cat. The most important finding is the dis- 
covery of two numerically important bundles of 
efferent fibers originating from higher auditory 
levels. One has its cells of origin in the nuclei of 
the lateral lemniscus, its fibers descend in the stria 
acoustic dorsalis to the dorsal cochlear nucleus of 
the opposite side. The other arises from the supe- 
rior olivary nucleus and terminates about cells of 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



the ventral cochlear nucleus of the same side. 
These efferents, plus the one previously described 
(1958), together constitute a rich feedback inner- 
vation from higher auditory centers. The fact 
that recent physiological experiments of several 
workers have shown that neural activity of the 
cochlear nucleus can be suppressed by stimulation 
of higher auditory regions leads one to assume that 
this phenomenon is effected through the descend- 
ing nervous connection described above. Dr. 
Gacek has played an important part in these in- 
vestigations particularly in performance of the 
operative procedures involving placement of the 
lesions visually. 

Two new features of the olivocochlear bundle 
have been revealed that the neurophysiologist must 
take into account. One is the presence of a homo- 
lateral component of the well-known crossed olivo- 
cochlear bundle; this arises from the S-shaped 
olivary nucleus and joins the crossed efferent coch- 
lear bundle before leaving the brain. The other 
finding is a connection of the olivocochlear bundle 
with cells of the anterior ventral cochlear nucleus. 

The study of efferent and afferent connection of 
higher auditory levels is being continued. 

Robert Boord, a student of the University of 
Maryland working here under a PHS predoctoral 
fellowship, completed his study concerning the 
question of possible presence of an efferent cochlear 
bundle in submammalian animals possessing a 
poorly differentiated hearing apparatus. The ex- 
perimental results demonstrate for the first time 
an efferent component of the cochlear nerve in the 
pigeon and alligator which is homologous to that 
found in mammals. The efferents have been traced 
as far as the receptor epithelium of the primitive 
organ of Corti and of the lagena, a structure of 
unknown function and not present in mammals. 
Knowledge of the presence of the efferents in 
animals having simpler organs of Corti than those 
of mammals is important relative to the deter- 
mination of the question of whether or not the re- 
ceptor cells have a dual innervation. It should 
be much easier to settle the question of ultimate 
termination of the efferents in an animal having a 
simply constructed receptor organ. This work is 
being used as a thesis for the degree of doctorate 
of philosophy. 

Dr. Gacek has pursued further the problem 
concerning; the ultimate termination of the efferent 



vestibular fibers. His attempts to solve this ques- 
tion utilizing various well-known experimental 
neuroanatomical techniques have proved fruitless. 
He has traced the efferents up to but not beyond 
the basement membrane of the receptor epithelium. 

Dr. G. Dohlman, internationally known for 
fundamental contributions on the anatomy and 
physiology of the vestibular receptors, joined the 
section in May and pursued histochemical studies 
on these organs. He plans to return from Europe 
near the beginning of next year to resume studies 
which concern determination of efferent innerva- 
tion of the receptors by histochemical method 
(cholinesterase activity) and the mechanism of 
stimulation of hair cells of the vestibular receptor 
areas. 

Mr. Kent Morest, a senior medical student of 
Yale, rejoined the Section under the COSTEP 
program for 2 months and completed the study 
begun last year on fiber connections of the area 
postrema. He will return July 1, 1960, as full- 
time investigator. 

SECTION ON PERINATAL PHYSIOLOGY 

During the past year the activity of this Section 
has been experimental investigation and extensive 
data collecting on adverse factors in the perinatal 
period of rhesus monkeys resulting in neurological 
and psychological deficits in the offspring. The 
first adverse factor tested was asphyxia neo- 
natorum. 

Data on menstruation in rhesus monkeys under 
standard conditions continue to be collected. 
Menstrual cycles of individual monkeys are sub- 
ject to wide variation. There has been no sys- 
tematic relation between regularity of cycles and 
fertility. Matings result in conceptions through- 
out the year. 

Data are being collected on maturation of rhesus 
monkeys. A nursery, patterned in many ways 
after those in hospital use, is maintained for the 
care of infant monkeys. Eecords are kept of daily 
weights, food intake, temperature, and respiratory 
rate. Heart rate is recorded electrically, grasp 
reflex is routinely measured ; and the maturation 
of the ability to self-feed is assessed. Normal 
ranges of values have been established. This 
knowledge has been applied to the care of infants 
damaged by asphyxia. 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



157 



Neurological deficits of experimentally induced 
asphyxia have been investigated. Monkeys of 
known mating dates were delivered by cae- 
sarean section near full term. Fetuses were 
asphyxiated by removing the uterine contents in- 
tact and waiting until intra- amniotic respiratory 
efforts ceased or were about to cease before free- 
ing the infant from the fetal membranes. As- 
phyxiation times were varied; some infants were 
able to breathe spontaneously, while others had 
to be resuscitated. Resuscitation was accomp- 
lished by intermittently inflating the lungs 
through an endotracheal tube. Activity, respira- 
tory effort, and heart rate were recorded. 

Some newborn monkeys were asphyxiated by 
placing them in a glass jar through which nitrogen 
was flowing. Other monkeys were delivered at 
once by caesarean section to serve as controls. 
Asphyxiated and control infants were raised in the 
laboratory and required the same constant nursing 
care as healthy and sick newborn human infants. 
Motion pictures were taken during the experi- 
ments and at intervals thereafter; neurological 
examinations were performed regularly; electro- 
encephalograph^ tracings were taken at intervals, 
and a great variety of physiological data were re- 
corded for later study, review, and comparison. 
Infants which seemed unlikely to survive, as well 
as some healthy ones, were killed by perfusion- 
fixation for histological studies. 

Technically satisfactory asphyxiation-resuscita- 
tion was occomplished in fetuses and newborns. 
The differences in the responses of fetal and new- 
born monkeys to asphyxiation observed during 
these studies fell into three groups : (a) the fetuses 
were quiescent except for respiratory efforts, 
whereas the newborns struggled, defecated, and 
salivated; (b) the heart rate fell slowly in the 
fetuses, while in the newborns the heart rate fell 
abruptly and then rose slowly; (c) the fetuses 
could be resuscitated 5!/2 minutes after their last 
gasps, whereas the newborns could not be resus- 
citated if asplryxiated for 1% minutes past their 
last gasps. These findings complement those of 
Dr. Dawes and his associates, working in this 
laboratory, who have shown in acute experiments 
differences in blood pressure, cardiac glycogen re- 
serve, and blood sugar between fetuses and new- 
borns asphyxiated past their last gasps. 



Standardized neurological examinations have 
been performed periodically from the time of 
birth. Motion picture records of the examinations 
are made at established periods, as are electroen- 
cephalographic recordings. Photic stimuli are 
used to activate the EEG. The neurological and 
electroencephalograph^ studies are carried out on 
both the asphyxiated and control animals. Neu- 
rological deficits of considerable degree and per- 
sistent nature have been observed in some of the 
asphyxiated monkeys. Within these, there has 
been little tendency for lateralized motor deficits. 
The general pattern has been one of choreo- 
athetoid movements and a marked lag in develop- 
ment of isolated movement, such as pawing, reach- 
ing, and picking up small objects. The EEG's of 
most of the asphyxiated animals did not differ 
greatly from the normal. The most marked find- 
ing has been a depression of the electrical activity 
during the first 5 or 6 post-asphyxial days. 

Miss Saxon has been studying the psychological 
effects produced by asphyxia neonatorum. Using 
both asphyxiates and normals in a test battery she 
has found no significant differences in learning 
ability between the groups so far tested (5 as- 
phyxiates ; 5 normals) . Also, no significant corre- 
lation was found to exist between the length of 
asphyxiation and object discrimination learning. 
However, normal controls have been found to be 
significantly more emotional than the asphyxiated 
subjects. Monkeys with severe neurological de- 
fects could not be subjected to these psychological 
tests. 

A year ago plans were initiated for a collabora- 
tive study between members of the field station 
Section on Perinatal Physiology and a group from 
the Nuffield Institute for Medical Research, Ox- 
ford, England. Dr. Geoffrey Dawes and three 
members of his staff from Oxford received a grant 
from United Cerebral Palsy Research and Educa- 
tional Foundation to permit travel to Puerto Rico 
for this work. They arrived in early August and 
conducted experiments on fetal physiology of the 
primate during August, September, and early 
October. Twenty monkeys were mated for this 
project. Eight infants were born spontaneously 
and 12 by cesarean section. Nine of the twelve 
mothers that were subjected to section survived. 
Several other newborn and young monkeys were 



158 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



made available for the study. Physiological and 
biochemical data were collected and are being 
readied for publication. Plans are being made to 
continue the research in Puerto Kico in another 9 
months. The most substantial findings are sum- 
marized in the following paragraphs : 

Observations have been made on 12 fetal 
monkeys in utero under pentobarbitone anaes- 
thesia, ranging in gestation age from 115-158 days 
(term is about 168 days). A leg was delivered 
through a small uterine incision and a catheter 
was inserted into the femoral artery for recording 
blood pressure and to obtain blood samples. In 
5 fetal monkeys, an arm was also delivered 
through a separate incision and the brachial artery 
was catheterized so that samples could be with- 
drawn from tributaries of the ascending and the 
descending aorta simultaneously. In the three 
youngest monkeys (126 days gestation or less) a 
uterine incision was made over the neck of the 
fetus and a catheter was introduced into the left 
carotid artery. 

The mean 2 saturation of the femoral arterial 
blood was 58 percent (range 51-63). Simulta- 
neous samples taken from a brachial artery were, 
on the average, 9 percent higher; values as high 
as 77 and 79 percent were observed. The blood- 
lactate concentration was of the same order as that 
seen in six monkeys, 8-12 days after birth (7-17 
mg/100 ml). In three fetal monkeys, femoral 
arterial blood samples withdrawn at the peak of 
a uterine contraction (producing a rise of intra- 
uterine pressure of up to 35 mm Hg) contained 
less 2 (1.5-9 percent saturation) than did 
samples withdrawn during a quiescent period be- 
tween contractions. On delivery of the fetus, the 
uterus contracted strongly and the arterial 2 
saturation fell to low values, comparable with 
those commonly observed in human-cord blood 
samples, even though the placenta was not yet 
separated. It is therefore suggested that cord 
blood samples, taken after delivery, may give a 
misleading impression of the conditions of intra- 
uterine life in the primate. And it is interesting 
that the same difference between the oxygen con- 
tent of the blood in the ascending and the descend- 
ing aorta exists as has been shown previously in 
ungulates. 

Dr. Combs, Dr. Dennery, and Miss Saxon have 
been carrying out electroanatomical studies on 



connections between the cerebellum, diencephalon, 
and cerebral cortex. These experiments were all 
carried out initially in cats, but similiar studies 
will be made on asphyxiated and control monkeys 
in order to seek neurophysiological evidence of 
damage through asphyxiation to this important 
motor pathway. "With electrical stimulation of 
the cerebellum, cortical responses were readily ob- 
tained from the contralateral anterior sigmoid 
(motor) gyrus. Smaller potentials were obtained 
from the contralateral posterior sigmoid gyrus. 
Ipsilateral responses, when present, were small, 
inconsistent, and only found in the anterior gyrus. 
The active cerebellar zone formed a longitudinal 
strip consisting of the lateral one-half of culmen, 
the medial two-thirds of crus I, the medial one- 
half of crus II, and the paramedian lobule. The 
largest response resulted from stimulating the an- 
terior two-thirds of the medial one-third of crus I. 
The results were as readily obtained in Nembutal- 
ized as in curarized preparations. 

In a related study, methodic stimulation 
throughout the diencephalon while recording 
from the ipsilateral sigmoid gyri has shown that 
the active diencephalic areas include essentially 
the sensory pathway and the course of the 
brachium conjunctivum. 

In 17 animals with prior mesencephalic destruc- 
tion of the medial lemniscus and the brachium 
conjunctivum, the active areas were the same with 
the exception of the anterior part of the red nu- 
cleus, the fields of Forel, the zona incerta, and the 
mesencephalic tegmentum. It has also been estab- 
lished that single-shock stimulation of the cerebel- 
lar hemispheres will induce in Nembutalized cats 
a bilateral multiple alpha-rhythm response in the 
cerebral cortex. This is most pronounced in the 
contralateral ectosylvian (auditory) cortex. 

An observational study of behavior and social 
organization of rhesus monkeys in the free-range 
colony on Cayo Santiago (now numbering 300) 
was begun in June 1956, by Mr. Altmann. Dr. 
Koford from the University of California is 
continuing these investigations. When the Labo- 
ratory assumed control of the Santiago colony, it 
consisted of about 150 monkeys. Since then, they 
have increased to nearly twice that number ; there 
are now about 280. The rate of mortality is low, 
about 5 percent per year. Five females have lived 
more than 20 years on the island. The ratio of 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



159 



mature males (5 or more years of age) to mature 
females (4 or more years of age) is 1 to 5. Mating 
activity commences late in July. Nearly all 
young are born during the period of 4 months 
commencing in February. Approximately 65 
percent of the mature females have infants born 
this year. At least since mid-1956, the monkey 
population has been divided into two principal 
social groups, one approximately twice as large 
as the other. Except for an occasional subadult 
male, members do not shift from one group to 
the other. The groups normally occupy separate 
parts of the island, though they share much com- 
mon ground. Near the center of each group are 
two or three of the largest males, which are clearly 
highest in dominance rank. Nearby are females 
with their young, up to 3 years old. Subadult 
males, low in dominance, are usually at the pe- 
riphery of the groups. 

Mr. Chandler, under the direction of Dr. Gavan 
at the Medical College of South Carolina, is carry- 
ing out anthropometric studies on the Cayo Santi- 
ago colony. They are conducting a longitudinal 
growth study with main emphasis on normal, de- 
velopment morphology. They hope to identify 
the normal rate, duration, and course of growth 
and to isolate some of the factors which may 
modify this pattern. 



OTHER ACTIVITIES 

The scientists of the Laboratory of Neuroana- 
tomical Sciences have been called upon to partici- 
pate in activities not directly related to conducting 
experiments. Several serve on committees and 
advisory panels. 

Dr. Palay transferred from the Anatomy and 
Physiology Fellowship Panel to the Cell Biology 
Study Section, DUG. He is also Secretary of the 
Assembly of Scientists of NIMH-NINDB. 

Dr. Lloyd Guth is a member of the Anatomy 
and Physiology Fellowship Panel, DUG. Dr. 
Milton Brightman is Vice President of the Wash- 
ington Society of Electron Microscopy. Dr. C. J. 
Bailey recently left this laboratory to serve as 
Executive Secretary of the Mental Health Study 
Section, DEG. 

Dr. Rasmussen serves on the Committee on 
Hearing and Bio-acoustics as a representative of 
NIH. 



Dr. C. M. Combs holds a courtesy appointment 
as Associate Professor of Anatomy, and Dr. H. N. 
Jacobson, as Associate in Obstetrics, at the Uni- 
versity of Puerto Rico School of Medicine. 
Neither one of these appointments carries teach- 
ing duties but both provide valuable contacts with 
other scientists. 

The chief of the Laboratory serves on the fol- 
lowing committees : Foreign Fellowship Commit- 
tee, DRG; Anatomical Sciences Training Com- 
mittee, DGMS ; Committee on Primates, National 
Academy of Sciences-National Research Council ; 
Executive Committee, American Association of 
Anatomists; Membership Committee, American 
Academy of Neurology; Committee on Interna- 
tional Collaboration, American Academy of 
Neurology; Research Advisory Board, United 
Cerebral Palsy Research and Education 
Foundation. 

Editorial tasks have engaged some of the in- 
vestigators' time during the year. The chief of 
the Laboratory is editor of Experimental Neu- 
rology ; Dr. Palay is on the editorial board of the 
same. Dr. Rasmussen is editor of a monograph 
entitled Neural Mechanisms of the Auditory and 
Vestibular Systems which is the sixth in a series 
of "Symposia in the Neuroanatomical Sciences," 
edited by the chief of the Laboratory. Dr. Guth 
is translator and editor of Ramon y Cajal's book 
on neurogenesis. 



Laboratory of Neurochemistry 

Until more space becomes available to the 
Laboratory, it will not be feasible to establish one 
or two desirable additional sections and to give 
more adequate working space to the two present 
Sections. Until such a time, we have had to 
abandon our search for a Laboratory chief. Dur- 
ing the year, we submitted plans which reflect 
our space and budgetary needs. We are hopeful 
that a new building will be constructed here to 
accommodate some of the growing needs of the 
Basic Research Program and that such construc- 
tion will be available for occupancy sometime 
during 1964. The Laboratory of Neurochemistry 
will at that time be due for major expansion. 
It is our plan in the meantime, after construction 
appropriations have been committed, to recruit 
and assemble the necessary personnel to make this 



160 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



a most effective and resourceful establishment for 
the investigation of neurochemical problems. 

Not a moment is to be lost in this enterprise be- 
cause advances in biochemistry and related 
achievements in other complementary mental and 
neurological disciplines are ripe for imaginative 
exploitation by a strong team of neurochemists. 
Although the Laboratory then will be much ex- 
panded and as yet only two elements of the total 
breadth of interest in this field are represented, it 
is clear from what we already have within the 
Laboratory and within the program that this ex- 
pansion of neurochemistry will represent growth 
from strength within this essential discipline. 

SECTION ON PHYSICAL CHEMISTRY 

Dr. David Davies has been carrying on further 
investigations of the formations and structures 
of complexes of macromolecules related to nucleic 
acids. Although the interaction of the synthetic 
polynucleotides, polyadenylate and polyuridylate, 
can lead to a complex which has the double helical 
configuration and paired complementary structure 
of native DNA, X-ray diffraction studies of the 
strongly aggregating system polycytidylate-poly- 
inosinate show no DNA -type structure. Centrifu- 
gal studies of this latter complex indicate strong 
aggregation, and spectral studies, moreover, show 
gross changes in both the rotational and vibra- 
tional frequencies associated with the purine and 
pyrimidine ring substituents of these molecules. 
The diffraction pattern of this simpler complex 
appears to be closely related to that obtained for 
the as yet undetermined structure of native DNA. 

Dr. Dan F. Bradley, with Dr. M. Kenneth Wolf 
of the Laboratory of Neuroanatomical Sciences, 
NINDB, Dr. Gary Felsenfeld of the University 
of Pittsburgh, and Dr. Audrey L. Stone, a visiting 
scientist in the Section, has been carrying out ex- 
tensive studies on the aggregation of dyes bound 
to polyanions. There exist a large number of 
cationic dyes which exhibit striking meta- 
chromatic color changes when used to stain poly- 
anions such as desoxyribonucleic acid, ribonucleic 
acid, heparin, and hyaluronic acid in tissue sec- 
tions. Perhaps the single most important dis- 
covery is that the structure of a polyanion deter- 
mines in part the strength of dye-dye interaction 
so that the color of bound dye can tell us about 



the polyanion to which it is bound. We have col- 
lected a considerable body of data which supports 
the generalization that the more rigid and well- 
ordered the polyanion, the weaker the dye-dye 
interaction. For example, all samples of native, 
well-ordered desoxyribonucleic acid (DNA) ex- 
amined show the same low value of this inter- 
action, but, when they are disordered by heat 
denaturation, the strength of the interaction in- 
creases. This observation has been developed to 
the point where the color of bound dye can be 
used to determine the degree of nativeness of DNA 
specimens. Applying this general principle to 
structural changes in synthetic nucleic acids it 
has been possible to confirm certain hypothesized 
transitions. A quantitative method of analysis in 
the microgram range for nucleic acids, mucopoly 
saccharides, and synthetic polyanions has been 
developed. 

Dr. David Davies and Dr. Sidney Bernhard, in 
collaboration with Dr. T. Viswanatha of the Lab- 
oratory of Pharmacology, NIAMD, and Dr. John 
C. Kendrew, of the MRC Unit in Molecular Bi- 
ology, Cambridge, England, have been attempting 
to establish the detailed molecular configuration 
of the atoms of an enzymatic site and it has been 
found possible thus far to crystallize some small 
enzymes and enzyme fragments. Some of these 
have been crystallized with specific substrates 
and/or heavy atoms. X-ray diffraction patterns 
of one heavy-atom labeled enzyme have been 
obtained. 

A new project this year is the study of enzyme 
models carried out by Dr. Sidney Bernhard with 
Drs. Ephraim Katchalski and Arieh Berger of 
the Weizmann Institute. Since the variety of 
enzyme-catalyzed reactions is enormous, it is neces- 
sary in any finite study to limit the models to 
either a particular enzyme or particular class of 
enzymes. In this investigation we have limited 
ourselves to the largest-known class of enzymes 
with a common catalytic amino-acid sequence. 
The sequence -glycyl-aspartyl-seryl-glycl- is cata- 
lytically inert. Derivatives of this sequence with 
the hydro xyl of serine esterified (a proposed en- 
zyme-substrate intermediate) show no unusual, 
reactive, chemical properties. Derivatives of this 
sequence in which the "beta" carboxyl group of 
aspartic acid is converted to an ester or an amide 
have striking properties. Thus, for example, the 






MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



161 



beta-benzyl-ester derivative of this sequence is 
hydrolyzed (debenzoylated) at a rate one million 
times faster than normal benzyl esters. The re- 
action has a "turnover rate" at neutral pH com- 
parable with enzymatic reactions. Molecular 
model constructions have resulted in a theoretical 
proposal of the three dimensional conformation 
of this molecule in the course of reaction. This 
"intermediate" conformation has been proposed 
as the catalytically active form of the enzyme, 
the configuration being stabilized by the protein 
superstructure. 

One of the most important concepts in molecu- 
lar biology is that the function of a protein is 
determined by the sequence in which the com- 
ponent amino acids are linked together. The first 
step in the theoretical approach to the problem 
of sequence determination in proteins was to de- 
velop a logical system for processing bits of in- 
formation about individual peptides to determine 
the unique sequence in the protein. Drs. Sidney 
Bernhard and Dan Bradley have developed such 
a system in collaboration with Dr. W. L. Duda 
at the IBM Company. We have provisionally 
named this system the Logical Unique-Sequence 
Tracer and have used it to reconstruct long se- 
quences (100 amino acids) from what seemed to 
be a hopeless confusion of data. However, the 
system cannot be used to its maximum efficiency by 
an individual because it involves a very large 
number of logical decisions so that it is being 
programed for a high-speed digital computer. 
With this program in operation, sequencing can 
be done with a minimum of experimental data. 
It is planned to carry out Gedanken fission experi- 
ments to find out on a statistical basis for real 
proteins how much information value is contained 
in various-sized peptides, the optimum size of 
peptides, value of determining only partial se- 
quences on peptides, etc. A preliminary result of 
great interest is that a method of "indifferent" 
fission in which a relatively large number of pep- 
tide bonds are broken with about equal velocities, 
e.g., by acid hydrolysis, is suggested as a method 
which might provide the optimum distribution 
of breaks in the protein to achieve maximum in- 
formation. We hope to test this suggestion ex- 
perimentally in conjunction with scientists in 
England who are working on the sequence of 
myoglobin. 



SECTION ON LIPID CHEMISTRY 

Dr. Eoscoe Brady has continued his investiga- 
tions on the pathways of synthesis of complex 
lipids. The report from this section in 1958 that 
malonyl coenzyme A is the key intermediate in 
the biosynthesis of long-chain fatty acids has been 
confirmed by two other groups of investigators. 
Work during the past year has dealt mainly with 
purification of the requisite enzyme system and 
the preparation of specifically labeled intermedi- 
ary compounds. A 600-fold purified enzyme 
system from rat liver tissue has been used to 
investigate the detailed mechanism of the condens- 
ing and reducing reactions required for fatty-acid 
synthesis. When the appropriate intermediary 
compounds are incubated with the enzyme, the 
only cofactor required for fatty-acid formation is 
a source of hydrogen atoms. Triphosphopyridine 
nucleotide (TPNH) is the preferred material al- 
though diphosphopyridine nucleotide is about 
two-thirds as effective as TPNH. 

Conversion of acetyl coenzyme A to malonyl 
coenzyme A was also discovered in this section in 
1958, and a report of this reaction was published 
early this year. This finding has also received 
confirmation in laboratories in the U.S. as well as 
Germany and Japan. The enzyme system has 
been purified, and the reaction exhibits the follow- 
ing dependence : There is an absolute requirement 
for adenosine triphosphate and the vitamin biotin. 
The presence of divalent metal ions is necessary, 
and the respective efficacy is Mg ++ >Mn ++ >Co ++ . 
A detailed study of the mechanism of this car- 
boxylation reaction is being carried out with a 
purified enzyme preparation. 

Dr. Eberhard Trams has worked on the forma- 
tion of complex sphingolipids which has resulted 
in the discovery that splenic tissue obtained from 
patients with Gaucher's disease catalyzes the for- 
mation of the accumulated offending cerebrosides 
in situ. With the use of various precursors of the 
cerebroside molecule, it has become apparent that 
the entire molecule may be synthesized de novo. 
These observations tend to render unlikely the 
suggestion that the etiology of Gaucher's disease 
is an excessive accumulation of catabolic materials 
from red-blood-cell destruction and is consistent 
with the failure of other investigators to demon- 
strate an increased level of plasma cerebroside in 
Gaucher's disease. 



162 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



We have prepared labeled psychosine (sphin- 
gosine-O-galactoside) for investigating the bio- 
synthesis of gangliosides, the polyhexose 
sphingolipids present in ganglion cells of the cor- 
tex. Preliminary experiments indicate that the 
enzymatic synthesis of these compounds has been 
successfully demonstrated for the first time in cell- 
free preparations of brain tissue. We have also 
devised an ultrasensitive analytical method for the 
quantitative determination of gangliosides. The 
availability of this procedure is required for 
studying the metabolism of gangliosides. We ex- 
pect to undertake investigations of this nature in 
cortical biopsy specimens obtained from patients 
with Tay-Sachs disease. This condition is charac- 
terized by the accumulation of abnormally large 
quantities of gangliosides in ganglion cells. 

A method has been devised by Drs. Eberhard 
Trams, Roscoe Brady, and Eugen Hecht for the 
quantitative determination of free sphingosine in 
plasma. The procedure was perfected in co- 
operation with Dr. Charles Sweeley of the Na- 
tional Heart Institute, and initial determinations 
indicate a level of 1 to 14 micrograms of sphingo- 
sine per milliliter of plasma. We expect to use 
this technique to determine the normal level of 
sphingosine in the cerebrospinal fluid and in 
samples obtained from patients with demyelinat- 
ing diseases. It has also been observed that free 
sphingosine can act as a prothrombin conversion 
factor. The significance of this finding on the 
mechanism of blood coagulation is under investi- 
gation. 

Within the past year, confirmation has appeared 
for the demonstration in this laboratory by Drs. 
Bernard Agranoff and Roscoe Brady of a new 
class of intermediary compounds called lipo- 
nucleotides. These highly reactive compounds are 
formed by the enzymatic reaction between phos- 
phatide acids (the monophosphate ester of a 
diglyceride) and cytidine nucleotides. Cytidine 
diphosphate diglyceride is the required intermedi- 
ate for inositol phosphatide formation. Inositol 
phosphatides exhibit very rapid metabolic turn- 
over in brain and are markedly affected by physio- 
logical and pharmacological agents such as 
acetylcholine and chlorpromazine. Inositol phos- 
phatides have been implicated in trans-membrane 
secretory processes, and the role of these materials 
in these reactions is under investigation. In the 



course of these studies, the nature of the metabolic 
antagonism between inositol and choline has been 
demonstrated. These substances compete with 
each other for the syntheses of essential phos- 
pholipids in growing animals. 

Dr. Bernard Agranoff spent the past year work- 
ing at the Max Planck Institut f iir Zellchemie in 
Munich where he participated in studies which 
dealt with several important steps in the pathway 
of the biosynthesis of terpenes. Specifically, he 
demonstrated the enzymatic isomerization of the 
5-carbon intermediate isopentenyl pyrophosphate 
to dimethylallyl pyrophosphate. The latter com- 
pound is required for the condensation of two 5- 
carbon fragments to form the 10-carbon geranyl 
pyrophosphate and subsequently the 15-carbon 
farnesol pyrophosphate. He participated in ex- 
periments which demonstrated the conversion of 
these larger molecules to squalene, the immediate 
precursor of cholesterol and other terpenoid ma- 
terials such as the steroid hormones and vitamins 
A, D, K, and E. 



Laboratory of Neurophysiology 

The Spinal Cord Section, under the leadership 
of Dr. Karl Krank, is proceeding with analysis of 
fundamental work on generation of impulses in 
nerve cells. The work is immediately aimed at 
determining what parts of the neuron produce the 
A and B spikes and analysis of the role of the 
dendritic processes. Clamping technics have been 
applied and the results are consistent with the pre- 
vious hypothesis that the A spike originates in the 
axon and that the B spike originates from mem- 
brane at least partly outside the soma. Work is 
going forward to plot the potential field around a 
single cell activated by antidromic stimulation. 
This project is dependent on application of some 
method of improving the signal-to-noise ratio. 

Practical methods for information-retrieving 
are under development in joint projects of this 
laboratory and the Laboratory of Clinical 
Sciences. A modification of a method devised by 
Mr. Robert Cox will be used by Dr. Frank in the 
first phase of this project. This is an important 
and pertinent area. Means must be found to raise 
signal-to-noise ratio for microelectrode work as 
well as for other purposes. 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



163 



Work is moving in the direction of analysis of 
integrating mechanisms of the neuron. 

It is generally assumed that CNS membrane 
potentials are sensitive to anoxia, and a recent 
publication purported to prove that assumption 
using intracellular recording technics. In a series 
of excellently designed experiments, Dr. Phillip 
Nelson, Dr. Frank, and Mrs. Mary Becker have 
shown this is not true. The stability of the mem- 
brane in hypoxia also has important consequences 
for certain aspects of spreading cortical depres- 
sion. Previous work has shown that, with macro- 
electrodes in the spinal grey, hypoxia produces a 
quick negative swing. The present work indicates 
this quick negative swing is essentially an artifact, 
probably due to abnormal sensitivity to hypoxia 
of the neurons injured by the electrode. 

The Section on Special Senses, under the lead- 
ership of Dr. Ichiji Tasaki, has several develop- 
ments. Perhaps the most important one is serious 
application of adequate theoretical treatment to 
tracer studies. This work was done in collabora- 
tion with Drs. Torsten Teorell from Uppsala, Ul- 
rich Franck from Darnstadt, Germany, and Leslie 
Nims from Brookhaven. It has been recognized 
for some time that tracer data often contain in- 
adequate information. Recently, rigorous theo- 
retical treatment of these problems has been ac- 
complished by Dr. Nims and others. Dr. Tasaki 
and his collaborators have extended these theo- 
retical developments and have made practical ap- 
plication of these concepts on both living and 
nonliving membranes, to get accurate information 
on ion movements. 

An extensive study of the movement of radio- 
active tracers across the squid axon membrane was 
carried out by Drs. Constantine Spyropoulos, 
Tasaki, and Teorell. The tracers examined in- 
clude Na 24 , K 42 , Ca 45 , tritiated water, CI 36 , labelled 
phosphate and sulfate. The data obtained were 
treated on the basis of the new concept developed 
by these investigators. 

The Section on Limbic Integration and Be- 
havior, under the leadership of Dr. Paul MacLean, 
has proceeded with a broad program in the gen- 
eral area of brain and behavior and the limbic 
system in particular. This program involves a 
comprehensive employment of behavioral observa- 
tions, conditioning and learning studies, electrical 



examination of the CNS, biochemical lesions, and 
neuroanatomical work. 

In the past there has been a notable lack of 
information about the localization of genital func- 
tion in the brain. The Section on Limbic Integra- 
tion and Behavior is employing the squirrel 
monkey in a systematic exploration of the brain 
to identify structures involved in penile erection. 
Thus far positive locus has been discovered within 
a major subdivision of the limbic system. 

Three other studies of a complemental nature 
are also in progress. One pertains to the function 
of the mammallary bodies. A second, conducted 
by Drs. John Gergen and MacLean, is aimed at 
analyses of the interaction between the highly ex- 
citable hippocampal system and other parts of the 
brain, as well as the interaction between the hip- 
pocampal system and associated neuro-endocrine 
mechanisms. The third pertains to a study of the 
sexual and social behavior of the squirrel monkey. 
Dr. Detlev Ploog is conducting, on a small group 
of squirrel monkeys, a valuable naturalistic study, 
which is remarkable not only because of the nature 
of the information obtained, but also as an illus- 
tration of what can be done with a small group of 
animals in a crowded laboratory setting. Dr. 
MacLean's introduction of the squirrel monkey 
into this laboratory has been in itself a valuable 
contribution, and it may have been partly instru- 
mental in introducing the squirrel monkey to the 
National Aeronautics and Space Administration. 
An anatomical atlas is being prepared to aid in 
the laboratory use of the squirrel monkey. The 
variety of work done by this section is unique, and 
is also a unique example of good basic science with 
closer than usual relation to clinical research. 

The Section on General Neurophysiology has 
been proceeding with several projects. One of 
these is an intensive program conducted by Drs. 
Eric Kandel, Alden Spencer, and Floyd Brinley, 
involving unitary extracellular and intracellular 
recording from the pyramidal cells of the hippo- 
campus of the cat. This work deals with the fun- 
damental excitation processes of the neuron. A 
great deal of new information has been obtained. 
Similarities with and differences from data 
derived from the spinal motoneuron have been 
demonstrated. The discovery of prominent de- 
polarizing after-potentials in hippocampal cells 
is particularly significant in view of the relatively 



164 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



high excitability of the hippocampus. It is inter- 
esting to note that the electronmicroscope pictures 
show a smaller than usual extracellular space for 
the hippocampal neurons. This supplies infer- 
ential support for the hypothesis that accumula- 
tion of K ion in extracellular space is the cause 
of the depolarizing after-potentials. There are 
also important inferences in this argument for 
mechanisms of spreading cortical depression. 
This program is gaining some knowledge of actual 
integrative mechanisms of which the firing of the 
cell axon is the end point. 

A valuable project, using tracers to examine K 
ion release from the cortex by various chemical 
agents and spreading cortical depression, was 
accomplished. 

Dr. Barbara Renkin is proceeding with an in- 
teresting method of conducting analysis of sensory 
discrimination at cortical and thalamic levels. 

Dr. Felix Strumwasser has made progress in the 
preliminary instrumentation of telemetering tech- 
niques to be used in studying manmmalian hiber- 
nation. He has made microelectrode studies of 
cells in brain and dorsal-root ganglion of the frog. 
Incidental to these studies a useful method was 
developed for extracellular stimulation of single 
cells permitting excitability studies to be made of 
units normally at rest. Dr. Strumwasser is apply- 
ing this technique to a study of excitability 
changes during some processes involved in learn- 
ing. Technical advances in recording from the 
dorsal-root ganglion of the frog have permitted a 
study of the relative roles of the different parts of 
the membranes of these cells. 

Dr. Freygang, working this past year with Dr. 
Richard Adrian and Mr. Andrew Huxley at the 
Physiological Laboratory, Cambridge, England, 
has been investigating the electrical characteristics 
of the membrane of muscle fiber with a special in- 
terest in properties that are peculiar to this kind 
of cell. Such properties need to be understood in 
order to relate the electrical events to the con- 
tractile properties of muscles. 

Laboratory of Cellular Pharmacology 

When the Laboratory of Cellular Pharma- 
cology was originated in 1954 a broad program 
of research was devised centering around three 
main lines of investigation: (1) biological meth- 
ylation, (2) amino acid metabolism, and (3) com- 



parative biochemistry. Although large overlaps 
have made subdivision of the various research 
activities among these different areas somewhat 
artificial, the device has nevertheless been useful 
in classifying our activities. 

While these areas continue to hold the interest 
of the laboratory, in the latter part of 1958 and 
more clearly 1959, some reorientation of the Lab- 
oratory's efforts took place, and the major areas 
of investigation can now be grouped more profit- 
ably around four topics: (a) mechanisms and 
pathways of protein biosynthesis, (b) biological 
methylation, (c) biological oxygenation and (d) 
alkaloid biosynthesis. These new areas stem more 
or less directly from the three original ones: for 
instance, alkaloid biosynthesis is conceptually re- 
lated to our lasting interest in comparative bio- 
chemistry and obviously studies on protein 
biosynthesis represent an extension of earlier 
efforts in the field of amino-acid metabolism. 

From an administrative standpoint, the most 
notable advance has been the establishment of a 
new Section on Alkaloid Biosynthesis and Plant 
Metabolism, and the inauguration of a new re- 
search program in this area. The research green- 
house facility has been in operation under Dr. S. 
H. Mudd since early spring and is now fully func- 
tional and adequately, if not yet optimally, staffed. 
More significant is the fact that the research pro- 
gram which had been originally formulated for 
this project and had formed the basis for its justi- 
fication and realization is already beginning to 
bear fruit, precisely along the lines we had 
anticipated. 

Our findings in this area illustrate once again 
the value of studying fundamental biochemical 
mechanisms in whatever biological material is 
most convenient with the assurance that the facts 
in a given form may well apply to widely diver- 
gent forms. 

Specifically, it has been established that, in the 
biosynthesis of the alkaloids, JST-methyltyramine, 
hordenine, and gramine, by cell-free extracts of 
barley or millet, the methyl group of these com- 
pounds is donated by S-adenosylmethionine. 
Moreover, it was shown that barley can indeed 
synthesize S-adenosylmethionine which is identi- 
cal to that made by vertebrates even to the extent 
of having the same stereochemical configuration 
about the asymmetric sulfur and a — carbon atoms. 



MENTAL HEALTH NEUROLOGICAL DISEASES AND BLINDNESS 



165 



Together, these facts very strongly suggest that 
the predominant pathway of plant transmethyl- 
ation lies through S-adenosylmethionine just as it 
does in vertebrates and microorganisms. 

A matter which requires further exploration is 
suggested by the structural resemblance of two of 
the particular alkaloids studied to the adrenal 
hormones and of the third to serotonin: If the 
role, as yet unknown, of these compounds in plant 
metabolism can be elucidated, perhaps we shall 
get a hint about the role of these neuro- 
hormones or related hallucinogenic materials. 

Another area of interest in regard to this sys- 
tem points up the particular advantages of plants 
as complex, biological forms, containing many 
specialized types of tissues and elaborate anatomi- 
cal and hormonal systems of intraindividual 
communication, and yet much simpler than the 
vertebrate and vastly more responsive to environ- 
mental manipulation and experimental control. 
Thus, the formation of the alkaloids now being 
studied is known to be under not only genetic 
control but under other controls as well, so that 
the formation occurs in a dramatic outburst at a 
specified stage of ontogenesis and in restricted 
types of tissues. It seems not unlikely that a 
study of the interplay of the control mechanisms 
at work here will give some insight into the im- 
portant question of how enzyme formation and 
activity are governed in higher organisms. The 
genetic, environmental, tissue-specific and hor- 
monal factors cooperating in this system are un- 
doubtedly complex but it is hoped that the great 
advantage offered by the relative malleability of 
the plant to experimental control will aid in 
elucidating these questions. 

With the realization of our plans in the alka- 
loid biosynthesis area it became desirable to di- 
vide the laboratory at least administratively into 
three sections under the leadership of Dr. 
Seymour Kaufman, Dr. Mudd, and myself. 

Dr. Kaufman's section continued to center its 
interest in the area of biological oxygenation. 
The two different enzymatic hydroxylation reac- 
tions being studied by Drs. Kaufman and 
Ephraim Levin are yielding results comple- 
mentary to each other in many ways. In both the 
phenylalanine and the DOPamine hydroxylating 
systems, new roles have been found for well- 
known vitamins — folic acid and ascorbic acid re- 



spectively. Specifically, it has been established 
that these cofactors play the role of electron 
donors in the reactions in which they respectively 
participate. In both systems, a substrate-de- 
pendent (i.e., phenylalanine or DOPamine) oxi- 
dation of the cofactors takes place resulting in the 
oxidation of tetrahydrofolic and to dihydrofolic 
acid in the phenylalanine system, and of ascorbic 
acid to dehydroascorbic acid in the DOPamine 
system. While the ultimate electron acceptor in 
both reactions is oxygen, it is possible that an 
enzyme-bound metal or even the substrate mole- 
cule may serve as the immediate electron acceptor. 

These studies, in addition to their intrinsic sci- 
entific value, contribute significantly to the area 
of basic research in the field of neurology and 
mental health for they are of obvious and direct 
significance to the problems of synthesis and 
function of the catecholamines, and to the patho- 
genesis of phenylketonuria oligophrenica. Indeed, 
the basic facts discovered by Dr. Kaufman in 
these studies are being utilized by Dr. Kaufman 
and Dr. William P. Weiss, of the Laboratory of 
Clinical Science, in a clinical study of phenyl- 
ketonuria children in the hope of discovering and 
devising some way to alleviate morbidity in this 
area. Although phenylketonuria contributes only 
a small fraction of the patients in the area of 
mental retardation it would be highly gratifying 
to be able to contribute in some measure to the 
solution of this medical problem. 

The work of the Section on Proteins centered 
primarily around two problems of fundamental 
interest. The protein-synthesis project, which is 
occupying the main interest of the laboratory, is 
a multipronged attack on what may be considered 
one of the central problems of biochemistry. At 
the present time, it appears most profitable to 
concentrate our efforts on a study of the chemistry, 
molecular configuration, and biological properties 
of S-RNA, a polynucleotide of relatively small 
molecular weight which appears to function as an 
acceptor and donor of activated amino acids for 
protein synthesis. S-RNA has been studied with 
respect to its physicochemical properties, as re- 
vealed by electrophoresis ultracentrifugation and 
viscosity measurements; its chemical nature, as 
demonstrated by base composition; and its enzy- 
matic and biochemical characteristics. A signifi- 
cant feature of the biological activity of S-RNA 



166 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



is its specificity for different amino acids but the 
basis for this specificity is yet unknown. Work 
from this laboratory has established that molecu- 
lar weight plays no role as a specificity determi- 
nant and furthermore that the different S-RNAs 
which are specific for the different amino acids 
all have similar or identical molecular weight. 
This suggests that the basis of the biological spec- 
ificity must reside in the nucleotide composition 
or sequence, and by a study of this problem it is 
hoped to obtain some clue as to principles of 
biological coding mechanisms. In these objectives 
the Laboratory has enjoyed the collaboration of 
Dr. Maxine Singer of the Laboratory of Bio- 
chemistry in NIAMD. 

The second problem (which has progressed fav- 
orably in the last year) has been the study of the 
properties of thetin homocysteine methylpherase. 
It has been known for some time now as the re- 
sult of the work of this laboratory that this pro- 
tein, which represents approximately 1 percent of 
the total liver protein, is capable of undergoing 
an interesting and rather unique reversible poly- 
merization reaction. The biological significance 
of this reaction is not yet fully understood; the 
possibility is under investigation that it may be 
related to the complex changes occurring during 
cell mytosis or even more generally that this pro- 
tein may fulfill some cyto architectural role in 
the cell. 

During 1959, the Laboratory enjoyed and bene- 
fited from the association of three visiting 
scientists : 

Dr. Olga Greengard, formerly of Middle- 
sex Medical School, London, England ; 

Dr. Othmar Gabriel, formerly of Vienna 
University and now at Columbia University ; 

Dr. Claude Blanc, formerly of Marseille 
University and now in the Laboratory of 
Cellular Pharmacology. 



Section on Technical Development 

During the calendar year 1959, the Section on 
Technical Development continued its role as a 
supporting organization. About 250 projects of 
various kinds were completed, serving the needs 
of virtually every section in each laboratory of the 
research program. Projects of representative 
types will be mentioned further on in this report. 



The Section has continued to be a source of 
material and components for investigators or their 
representatives. Procurement is carried out with 
the needs of the entire basic research program in 
mind. Obsolete equipment and components have 
been shunted to Surplus Property in order to bet- 
ter utilize the available space for an up-to-date 
stock. The blanket purchase-order arrangement 
with Capitol Radio Wholesalers has proven itself 
so well, due to complete inventory and fine co- 
operation, that it is now possible to maintain 
adequate stock in the Section on Technical De- 
velopment at the expense of only a half rather 
than a whole module of space. This provides one 
additional work area and makes practical the re- 
cruitment of another technician. 

Addition of Mr. Paul Byrne, a general labora- 
tory mechanic, to the staff during 1959 resulted in 
a very significant decrease in the waiting time of 
the researchers who have a purely mechanical 
problem, and permitted detaching one man for 
thirty days' duty at the Marine Biological Labora- 
tory, Woods Hole, Mass. 

Additional power supplies, waveform gener- 
ators, and other universally used instruments have 
been made available, on loan, for short-term ex- 
periments or as interim equipment during purchas- 
ing delays. 

The Section's fund of technical information and 
new product listings is being improved continu- 
ally. As new equipment and components are de- 
veloped, literature is requested and filed. A tech- 
nical library is slowly and carefully being built, 
with some 60 volumes of text on physics, elec- 
tronics, semiconductors, optics, and biological 
techniques now on hand. 

The Section's time is divided among several 
functions: counsel, construction, development, 
maintenance, and repair. Each category is a part 
of both internal operation and services to the 
designated Laboratories. Time permitting, ideas 
originating within the Section are implemented, 
where they will be of value in the future, although 
not yet the object of a specific request. The ma- 
jority of time is spent in the construction of new 
instruments and devices to specifications pre- 
sented by investigators. Equipment previously 
used is adapted or modified for another purpose. 
Repairs are made on both commercial equipment 
and the specialized units that have been developed 
at the NINDB or NIMH. 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



167 



Working space and direct assistance are still 
provided to the scientist who wishes to work on a 
problem himself. This continues to be a much 
used and much appreciated function of the Sec- 
tion. It also seems to fit in well with projects 
underway by members of the Section. 

The goals of the Section on Technical Develop- 
ment are unchanged. Foremost is the develop- 
ment of a better tool for the scientist to use, in an 
effort to create the means for the realization of an 
idea. As thinking extends a tool to its limits, the 
justification and need arise for another and better 
tool. Secondly is the concept of direct assistance 
whenever possible — assistance meaning another 
pair of hands in the scientist's own laboratory, the 
loan of equipment and components, and the ex- 
change of ideas. 

In regard to projects completed, a resume of 
several representative types will be given. 

An auxiliary cathode-ray tube display system 
was designed and built. This "slave scope" works 
with the Tektronix Type 502, a dual-channel oscil- 
loscope. Waveforms may be viewed on either 
channel of the "slave" simultaneously with the 
same waveform on the corresponding beam of the 
master. Adjustments are provided to equalize 
such variables as intensity, focus, sensitivity etc. 
It is used in conjunction with a camera, enabling 
permanent records to be made. Another slave 
system, using the Tektronix Type 535 Oscilloscope 
was built. It is similar to previous models using 
the Type 535, the variation being one of control 
circuitry. 

It seemed advisable to develop a power supply 
capable of powering a Type 122 pre-amplifier, 
which normally uses batteries as a power source. 
Due to the expense of batteries and their inoppor- 
tune failures, a supply was designed and built, 
using plug-dn stages. Later, due to an increase in 
cost of these units, it was built without the plug-in 
units. A third model now on the design boards 
will make use of the regulating capabilities of 
zener diodes. This model should be useful as a 
battery replacement for the more demanding d.c. 
amplifiers. 



Design and construction of a working model of 
an electrode holder and advance mechanism are 
now complete. With this unit, dual concentric 
micropipettes can be accurately positioned and 
the inner controlled with respect to the outer. Not 
only is this a critical mechanical assembly, but 
provisions had to be made for electrical connec- 
tions and for mounting to the amplifier in such 
a way that removal can be rapid. Negotiations 
are completed for the production of a small num- 
ber of these mechanisms by a private concern. 

A device for the control and programing of 
light stimuli has been developed for experimenta- 
tion with rats. By means of programing the light 
patterns, apparent movement can be simulated. 
This apparent movement can be regulated with 
respect to time. The effect of the moving stimuli 
upon the animals is then observed and recorded. 

Several high-input impedance probes have been 
constructed with miniaturization, shielding, and 
ease of operation as considerations. The input 
electron tube is mounted in especially machined 
aluminum shields, turned down to a few mils in 
thickness to reduce size and weight. Work is now 
progressing towards design of a probe unit to be 
compatible with the electrode holder and advance 
mechanism mentioned previously. 

An intercom has been designed for use between 
an observer and two subjects, all located in sep- 
arate rooms. Signals, as well as verbal communi- 
cation, are possible with this system to be used in 
dream studies in conjunction with an EEG re- 
corder modified for this purpose. Design and con- 
struction of amplifiers and signal oscillators are 
complete. Further EEG modifications are 
pending. 

Additional enumeration probably serves no pur- 
pose since a complete list cannot be presented in a 
report of reasonable length. The projects are 
widely varied, including repair and modification 
as well as other new equipment. The coming year 
will show further progress along the lines of more 
and better service to the research program. This 
progress will be due to the increasing proficiency 
of the present staff and, hopefully, to the addition 
of another capable electronics person. 



168 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



CLITSICAL INVESTIGATIONS— NIMH 

INTRODUCTION* 

The inception of the clinical investigations pro- 
gram seven years ago afforded an unusual oppor- 
tunity for the development of a mental-health 
research unit. Eighty beds were made available, 
together with ample nursing, social service, and 
physical and recreational therapy support. Since 
the beds were in a Clinical Center rather than in 
a hospital, there were no service obligations, no 
teaching responsibilities. In fact, the beds did not 
even have to be devoted to patient care, but could 
be used for a variety of studies of normal indi- 
viduals. There was similar freedom with respect 
to recruitment of investigators. At a time when 
research foundations were beginning to consider 
the merits of block versus project research grants, 
and when the idea of lifetime appointments for a 
limited number of research professors was just 
being broached, we were able to select several life- 
time laboratory chiefs, a number of section chiefs, 
and to provide each of them with block support 
for long-range studies of broad scope. 

In these circumstances it seems appropriate to 
scrutinize carefully the established psychiatric re- 
search organizations. Did the freedom from con- 
ventional teaching and service responsibilities and 
reasonable assurance of long-term support afford 
us an opportunity to study new problems or old 
problems in new ways? Should we endeavor to 
utilize these resources to establish a more or less 
traditional setting hopefully free, perhaps, from 
some of the common practical and material bur- 
dens which restrict freedom for research? Or 
should we to the best of our abilities try to de- 
velop a different type of setting and organization 
which might facilitate the production of data 
which could augment and extend the significance 
of the important studies being carried out in many 
excellent service and teaching hospitals? 

It was felt that among the more important but 
least amply supported (by systematic observation) 
theories in psychiatry were those related to de- 
velopmental and psychosocial determinants of 
personality formation and behavior. The great 



•Prepared by Robert A. Cohen, M.D., Ph. D., Director of 
Clinical Investigations, NIMH. 



concentration of biological scientists at the Na- 
tional Institutes of Health promised powerful 
support for investigations of the organic bases of 
normal and abnormal behavior. At the same time 
the location of our facilities in a structure which 
was architecturally designed and administratively 
geared for an organic approach to chronic disease 
dictated against the establishment of a community 
health facility with in- and out-patient, consulta- 
tive, and home-visit services, which might also 
have provided a unique setting for significant 
mental-health studies. It was decided, then, to 
make the study of behavior rather than psychia- 
tric treatment our central theme, and to study nor- 
mal behavior as well as a selected variety of 
behavior disorders. This did not mean that treat- 
ment would be neglected ; on the contrary, it was 
hoped to establish a highly effective therapeutic 
setting. The openness and freedom of expression 
which occur in successful therapy provide infor- 
mation about thoughts and feelings which cannot 
be gained by other means. The study of therapy 
brings to light some of the important forces which 
operate to change behavior patterns. This has 
obvious implications for education, and for any 
culture which would hope to bring about a fuller 
and more creative life. Similarly, to the extent 
that mental illness can be looked upon as an ex- 
periment of nature, the study of the social, psycho- 
logical, genetic, and biological variables which 
may appear in consistent patterns with different 
types of personalities and disorders would provide 
the basis for a more powerful theory of behavior 
than is now available. The establishment of longi- 
tudinal studies of child development would pro- 
vide an ideal instrument for verification of 
hypotheses derived from cross-sectional ap- 
proaches, and would in turn feed back suggestions 
for scrutiny by the groups studying other phases 
of behavior. Finally, the opportunity of setting 
up a clinical neuropharmacology center in col- 
laboration with Saint Elizabeths Hospital would 
significantly extend the scope of the experimental 
studies in which we could engage. 

In keeping with this broad goal we did not limit 
our recruitment to the clinical therapeutic dis- 
ciplines, but sought anthropologists, sociologists, 
and psychologists on the one hand; pharmacolo- 
gists, physiologists, and biochemists on the other. 






MENTAL HEALTH NEUROLOGICAL DISEASES AND BLINDNESS 



169 



The range of disciplines and the areas of study 
are roughly indicated in the table below : 





Pathological behavior 


Normal behavior 


Animal 

be- 
havior 


"Acting 
out" 
dis- 
order 


Dis- 
orders 

of 

thought 

and 

affect 


Psycho- 
somatic 

dis- 
orders 


Adult 


Aged 


Child 


Anthropology 

Sociology 

Psychology 

Psychiatry 

Pharmacology 

Physiology 

Biochemistry 















Not all these areas are being intensively studied, 
nor is each discipline represented in each study. 
In their individual reports, the laboratory and 
branch chiefs have given substantive reviews of 
the work in progress. Before proceeding to them, 
I wish to point out some of the issues we have en- 
countered in our efforts to give substance to this 
idea for a mental-health research program. 

The first problem faced was that of staff recruit- 
ment. For such a goal as has been outlined, it 
would have been desirable to have brought to- 
gether laboratory chiefs representing at least 
several of the important disciplines, to have given 
them time to get acquainted with each other's con- 
cepts, to outline several investigative problems 
of mutual interest in addition to others they would 
pursue individually, and then to assemble the 
supporting staff. Even in this demiparadise, 
however, time was a precious commodity. A com- 
mitment had already been given to open the first 
ward, 4% months after the Director of Clinical 
Investigations was appointed, and by the end of 
the year a 50-bed unit was supposed to be in opera- 
tion. Although this time schedule was not strictly 
enforced, it is not necessary to stretch one's imagi- 
nation to picture some of the difficulties en- 
countered. Both the investigative groups and 
the hospital organization were built up from 
scratch. Every appointment had to be made 
through the U.S. Public Health Service Commis- 
sioned Corps or through the Civil Service system. 
And when finally a staff was assembled, each mem- 
ber bringing with him the point of view and way 
of operating which were traditional at his previous 
assignment, we had to forge a new set of pro- 



cedures which would be suitable for our goals, for 
our staff as a whole, and for our new setting. 

Since it was not possible to recruit the labora- 
tory chiefs and bring them together to plan our 
joint program before operations actually began, 
the directors of each of the major divisions were 
appointed as soon as the interest of a suitable in- 
dividual could be enlisted. Each laboratory chief 
joined in the consideration of those who were ap- 
pointed subsequently, which has made possible 
good personal as well as potentially good intellec- 
tual and working relationships. This, in my 
opinion, is relevant to the effectiveness of inter- 
disciplinary research since I believe that some con- 
ceptualizations may require more than one mind, 
more than one way of looking at behavioral events. 
However, the result of this manner of program 
growth has been the development by each labora- 
tory and branch chief first of his own individual 
program. It took 5 years to bring the group to- 
gether, and now after 7 years, I believe that it 
would be fair to say that each laboratory has de- 
fined its major research goals; in each of them 
creditable investigations are well under way, and 
some significant studies have already been com- 
pleted. In individual projects there has been con- 
siderable collaboration between laboratories, and 
in some of the laboratories several disciplines are 
strongly represented. In addition, there is a 
degree of overlapping interest between labora- 
tories in several important research areas, even 
though each group varies its approach and focus. 
All this could serve to support a coalescence of in- 
terest in one or two problems of major theoretical 
importance which might constitute a limited por- 
tion of each laboratory program. While many 
factors favor the gradual formulation of several 
such broad research studies, it remains to be seen 
whether they are all that is necessary for truly 
creative interdisciplinary work. 

I believe the first phase of the development of 
our research program is over. We are no longer 
laboring just to secure a firm footing, but can turn 
more of our attention to the circumstances under 
which we work. There are two sets of condi- 
tions — one external, the other internal — which 
affect our creativity, our sense of satisfaction and 
fulfillment in our work, and, consequently, our 
productivity 






170 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



The National Institutes of Health developed 
from the old U.S. Public Health Service Hygienic 
Laboratory, and the list of our forebears includes 
such distinguished names as Rosenau, Reid Hunt, 
Voegtlin, Stiles, Mansfield Clark, Goldberger, 
Francis, and, more recently Romberg. Despite 
this excellent record, every senior investigator we 
have approached in our recruitment program has 
had serious questions about freedom of research in 
a Government institution. Where public money is 
spent, controls of some sort are invariably insti- 
tuted, questions asked at frequent intervals, certain 
regulations observed. Research workers tradi- 
tionally believe that regulation should come from 
within themselves, and not imposed by external 
authority. Personal contact is important to them, 
and they are concerned about working in an organ- 
ization whose very size makes it necessary that 
some decisions affecting their work be made at 
levels several steps removed from them. Those of 
us who look upon our land as the cradle of liberty, 
must feel some concern at the fairly widespread 
belief among scientists that, as liberty has become 
institutionalized, and scientific freedom restricted, 
a Government appointment should not be seriously 
considered. 

On the other hand, however, there are many who 
believe the Government has a responsibility to 
conduct basic research, and are interested in be- 
coming personally involved in the development of 
what they believe can become an excellent research 
institution. In my opinion we have made notable 
progress toward that goal. At the bench level, 
research problems can be selected by the investi- 
gators who will work on them. Unless new and 
additional space, equipment, and/or personnel are 
sought, the investigators have a high degree of 
control over the resources which will be employed 
in the attack upon the problem. As I see it, the 
most serious and difficult question which confronts 
us is that of program evaluation. This regularly 
comes up when promotions are considered and 
when proposals for expansion are presented. It 
becomes an issue when any request is made calling 
for a break in routine and established procedures. 
It is generally accepted as desirable to have strong 
representation of related scientific disciplines in 
each of our major program areas. It is obvious to 
everyone that in the large population recruited as 
a result of this decision, there must be variations 



in the competence and creativity of the research 
staff — some may approach genius, others of us may 
never be more than pedestrian. It is equally obvi- 
ous that as the program expands and as the num- 
bers of scientists increase, decision making can 
no longer be confined to the relatively small group 
who had the vision and who have borne the re- 
sponsibility for the development of the National 
Institutes of Health as they stand today. 

The Government operates as a line organization 
however, and line organizations make no provision 
for delegation of decision-making responsibility. 
The man on top remains responsible. As program 
grows and numbers increase, he has less and less 
contact with the individual members of his staff. 
Administrative routines are established to deal 
with regularly recurring and commonly accepted 
procedures. But the break in routine calls for 
individual consideration and makes a demand 
upon the chief's limited time. He no longer is 
able to maintain his earlier personal involvement 
in each phase of the developing program. There- 
fore he sets up a number of intermediate persons 
or groups to review requests and to advise him 
concerning the appropriate decision. In fact, if 
the program grows large enough, he may even call 
upon outside groups to assure him that the pro- 
posed plan has been carefully considered and 
merits support. In this process what begins as a 
highly personalized research issue or problem or 
request becomes progressively depersonalized as 
it passes from level to level on its way through the 
decision-making machinery. Such circumstances 
are obviously not conducive to creative effort, 
particularly when — as sometimes but not always 
happens — the effort may constitute an assault 
upon cherished concepts. It is here that the prob- 
lem of individual creativity enters the picture. 
Most of us would grant an Einstein, a Fermi, 
or a Darwin any condition he deserves, but is 
equal freedom to be granted to the mine-run 
investigator ? My answer to this question would 
be emphatically "Yes!" There is considerable 
evidence to support the belief that creativity 
is fostered by assigning the decision-making re- 
sponsibility to the level at which all the immedi- 
ately relevant facts are available. The develop- 
ment of the Assembly of Scientists is a step in 
this direction ; the more the scientists as a group 
can share in the decision-making process, the more 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



171 



responsive will it be to their needs as productive 
investigators. 

But the most important answers to the question 
of what is needed to do thoughtful research lie 
not in our stars but within ourselves. It has, 
perhaps, been more stressful than we realize to 
come to this new type of institution and to settle 
down immediately to the pursuit of new knowl- 
edge. If a group from Walla-Walla should be 
first to settle on the moon, those of us who arrived 
a year later might expect to find a number of 
institutions closely resembling those we know exist 
in Walla- Walla. In fact, the very names of New 
Orleans, New York, New Haven, and New London 
suggest that for ages past the thoughts of ex- 
plorers turn toward the scenes of their childhood. 
Although it is true that research was fostered first 
by the learned societies, it soon found its home 
in the universities where, until recent years, it 
largely remained. Although universities were 
supported by lordly patrons, research could hardly 
be said to have lived a life of luxury. Tradi- 
tionally, it was conducted in a garret, with simple, 
home-made equipment, at odd hours snatched from 
teaching and family responsibilities, and often 
drew its inspiration from the pressure of conflict- 
ing forces with which the teacher or doctor was 
wrestling in the course of his daily life. In some 
instances the researcher did not even have the 
blessing of a university position — Einstein worked 
in a patent office, Beaumont at an isolated Army 
post. Probably such events as the successful de- 
velopment of the atomic bomb and Ehrlich's dis- 
covery of the effectiveness of 606 gave support to 
some of the thinking which led to the building of 
the Clinical Center. After all, if Einstein de- 
veloped the theory of relativity in a patent office, 
what greater ideas might he have had if he had 
worked from the beginning in a large research 
institute? If Ehrlich had tested the therapeutic 
effectiveness of all 606 of his preparations simul- 
taneously, might not millions of people have been 
spared the ravages of syphilis? If Shakespeare 
composed his poetry for a group of ragged play- 
ers, what beautiful images might he not have 
brought forth if he had written from the comfort 
of an endowed chair ? 

Most of us came from universities and hospitals 
where we had to teach and had to treat patients. 
We had longed for an easing of these burdens, 



for time to pursue our ideas in leisurely and 
thoughtful fashion. But few of us ever dreamed 
that we would one day be given an institution not 
only with freedom from but an injunction against 
teaching and service, and told simply "create!" 
Eesearch ideas come in unplanned-for places and 
at unplanned-for times, and not necessarily most 
often when one's professional life depends upon 
their appearance. It took an environment such 
as NIH which, despite its limitations, has, per- 
haps, the greatest degree of academic freedom to 
be found and makes us realize how truly pro- 
tective teaching and service may be. When one 
earns his bread by these means, his research be- 
comes a delightful avocation which can only serve 
to increase his self-esteem. Under such circum- 
stances one dares to be unconventional, to let 
fancy roam — and perhaps it is no accident that 
basic research has flourished in universities more 
than it has in industrial laboratories, the occa- 
sional notable exception only serving to prove the 
rule. The fact is that NIH does resemble a 
foundation for industrial research in that our suc- 
cess is measured by a product alone, and not only 
by teaching and service as might be true in a uni- 
versity. Even though the product is basic re- 
search itself and not a better way to make 
lipstick, the concentration upon it and exclusive 
dedication to it might conceivably impede prog- 
ress under certain circumstances. The atomic 
bomb and Ehrlich's final discovery of 606 were 
engineering feats. A basic-research institute can- 
not be organized for that type of activity. 

Whatever the correct interpretation may be, I 
have seen the new staff member, after his first 
breath of freedom, begin to look around him for 
the chains he laid aside. Invitations to lecture 
are eagerly accepted. Some of us feel that we get 
our best ideas while teaching a group of eager, 
dedicated graduate students ; others, while we are 
wrestling with the responsibilities of treating 
desperately ill patients. We find ourselves long- 
ing for some of the university structure and 
atmosphere. Still others find themselves at times 
wanting more colleagues to work with them; 
somehow, in order to seem truly worth while, the 
program in which they engage should cover every 
conceivable facet of the research problem in ques- 
tion. I do not mean by this remark to cast doubt 
on the value of exhaustive attacks upon important 



172 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Middleton, Chief of the Psychiatric Nursing Serv- 
ice, and her staff, in meeting the many problems in 
connection with the nursing care of the patients, 
has contributed immeasurably to the development 
of our Clinical Investigations program. 

The clinical service of the Child Eesearch 
Branch was discontinued June 30, 1959, with the 
termination of the clinical studies of that Branch. 
The clinical facilities used by the Child Eesearch 
Branch were transferred : Nursing Unit 4-E to the 
Adult Psychiatry Branch, and Building T-4 to the 
Biosocial Growth Center. 

The Biosocial Growth Center, administratively 
a unit of the Office of the Director of Clinical In- 
vestigations, NIMH, began operations July 1, 
1959, under the direction of Dr. Wells Goodrich. 
This new program is designed to investigate the 
earliest influences on normal growth, through con- 
current studies of parental attitudes in prospective 
parents with a group of newly married couples, 
parents of newborn, and parents of healthy 2-year- 
old boys. At the same time, studies of neonates 
are being carried out in collaborating hospitals, 
and of normal 2-year-old boys in a nursery school 
setting in Building T^. The research in Build- 
ing T-4 is carried out as an out-patient operation 
through the Admission and Follow-Up Depart- 
ment of the Clinical Center. 

In the past we have obtained the recommenda- 
tion of the Medical Board and the approval of the 
Director of NIH for some of our psychological 
studies with normal subjects on an ad hoc basis. 
Recently, greater recognition was given to the 
NIMH need to carry out certain psychological and 
sociological studies in which the usually required 
medical work-up is not indicated or is contraindi- 
cated. The Director, NIH, has approved the 
Medical Board recommendation for an amend- 
ment to the Organization and By-Laws of the 
Medical Staff of the Clinical Center, regarding 
psychological and sociological studies in Building 
T-4. This is a step in the direction of facilitating 
approval procedures for psychosocial studies of 
normal subjects. 

On several of our units we have patients and 
normal subjects who remain here for long periods. 
It is therapeutically desirable to provide work op- 
portunities for these people, consistent with their 
clinical status and the treatment goals for them. 

♦Prepared by William C. Jenkins, M.D., Chief of Clinical Care. We have had Some SUCCeSS in finding Work for 



problems, but simply to point out that on occa- 
sion we may have an impulse to assuage unwit- 
ting anxiety about the merit of a more-or-less 
limited problem by expanding its limits as if we 
were thereby increasing both its importance and 
its merit. 

These, as I see them, are some of the important 
problems which face us in our effort to develop to 
its fullest the remarkable opportunity afforded at 
NIH. For this new type of institution we shall 
have to build, as the universities did over a longer 
period of time, structures which foster the highest 
degree of creative thought on the part of bench 
scientists and administrative leaders alike — 
structures not necessarily modeled precisely after 
those which have proved successful in universi- 
ties, but which are fashioned out of the unique 
attributes of our own situation. 



Report of the Chief of Clinical Care* 

During the past year we consolidated our clini- 
cal care activities into two services. The Adult 
Psychiatry Branch service has three nursing units 
providing the settings for the study of family re- 
lations in schizophrenia, for the study of first-year 
college students who develop a psychiatric illness 
requiring hospitalization, and for psychosomatic 
studies with a group of normal control subjects. 
The Clinical Science Laboratory service, with two 
nursing units, uses one for a group of male chronic 
schizophrenic patients in the study of biological 
factors in schizophrenia, and the other for a group 
of normal subjects who serve as controls. Each 
unit is administered with considerable autonomy, 
integrated with the total program of the respec- 
tive clinical service involved, and the services co- 
ordinated within the framework of policy and 
practice for care of patients within the Clinical 
Center. 

The Psychiatric Nursing Service has been or- 
ganized along the same lines. Interchange of 
nursing staff personnel between the nursing units 
of a service has facilitated development of greater 
understanding of the total research activities of 
the service, resulting in more interest and effective- 
ness in carrying out the work. The enlightened 
and devoted interest and support of Miss Agnes 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



173 



some of them but a wider range of such activity 
is needed. We are continuing our efforts to find 
a more favorable solution to this problem. 



Adult Psychiatry Branch* 

The Adult Psychiatry Branch has undergone 
major changes in the past 2 years. During 1958 
there was extensive recruiting of new staff mem- 
bers, reorganization of existing projects, remodel- 
ing of space, obtaining of equipment, and gen- 
eral tooling up for the planned new research. In 
1959, data collection got underway in several new 
lines of inquiry. A tangible sign of change dur- 
ing the year was the opening of two new research 
units in the hospital (3-East and 4-East). In 
January we began admitting as patients to 3-East 
college freshmen from a variety of eastern schools 
who encountered serious difficulty on going away 
to college, and in June we admitted to 4-East our 
first group of healthy young volunteers for psy- 
chophysiologic research. This latter unit makes 
possible a degree of close observation rarely pos- 
sible in psychosomatic studies. In addition, it 
permits utilization of environmental conditions 
for experimental purposes, and brings our various 
psychophysiologic woi-kers — necessarily an inter- 
disciplinary group — into close contact with each 
other. 

In our Psychosomatic Section we are attempt- 
ing to determine the extent to which the day-to-day 
interactions of the human organism with its en- 
vironment are reflected in endocrine function. Re- 
search in recent years has shown the extent of 
CNS regulation of endocrine function and, con- 
sequently, a wide range of visceral functions. Our 
research is attempting to determine whether the 
emotional fluctuations of everyday living are asso- 
ciated with concomitant fluctuations in concentra- 
tion of hormones that have widespread physio- 
logical significance. In this connection, we would 
like to quote from a chapter by our close collabora- 
tor, Dr. John Mason, in the 1959 Annual Review 
of Physiology : 

* * * It appears in general that the importance of 
central nervous system influences upon visceral func- 
tions, particularly endocrine regulation, has not been 
fully appreciated, probably for a number of reasons. 

In the first place, there has been the rather well- 



•Prepared by the Chief, David A. Hamburg, M.D. 



established general impression, supported by experi- 
mental data, that visceral functions are largely auto- 
nomous and responsive primarily to metabolic needs. 
The existence of these plausible concepts of self- 
regulation has possibly created resistance to the idea 
than an additional set of regulatory influences of 
considerable practical importance might be exerted 
upon visceral functions by the central nervous system. 

Probably a more important reason, however, has 
been the crucial matter of experimental approaches 
and methods. It is clear, first of all, that a compre- 
hensive experimental approach to neural regulation 
of visceral function can only be achieved by the pool- 
ing of skills and viewpoints of a broad range of 
scientific disciplines. These combined approaches 
must permit, on the one hand, an analysis of central 
nervous system mechanisms (by the techniques of 
neuroanatomy, neurophysiology, neuropharmacology, 
neurochemistry, experimental and clinical psychology, 
and psychiatry) and, on the other hand, the analysis 
of specific visceral functions (by the techniques of 
chemistry, physiology, pathology, immunology, and 
internal medicine). Impetus for collaboration be- 
tween various combinations of disciplines within these 
two general groups has come from both directions. 
Many behavioral scientists have come to view hope- 
fully the measurements of visceral function, which 
are relatively objective and quantitative, as a major 
approach to the experimental analysis of emotional 
states, which have been extraordinarily difficult to 
evaluate by psychological techniques alone. Sim- 
ilarly, physiologists and internists have become in- 
creasingly aware of the necessity for a greater under- 
standing of the impact of emotional disturbances upon 
bodily function and of the possible participation of 
emotional factors in the development of disease. 

Within the past 5 to 10 years substantial new 
methodological developments have emerged in many 
fields and have made possible a new order of direct- 
ness and accuracy in psychophysiological research. 

A typical problem in research of this sort is the 
difficulty in utilizing the best available chemical 
methods for hormone measurement. The active 
participation of Dr. Mason and his colleagues pro- 
vided microanalytic techniques that permit reli- 
able detection of small differences. 

Another typical problem in this kind of research 
is the difficulty of obtaining reliably observable 
emotional responses for experimental purposes. 
This is sometimes done by observing naturally oc- 
curring stressful situations and sometimes by ex- 
perimentally devised situations. These approaches 
tend to complement each other and we are attempt- 
ing to use both. 

Research on human behavior and adrenocortical 
function in recent years has established the fol- 
lowing points: (1) Plasma and urinary hydro- 



174 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



cortisone elevations occur in circumstances of dis- 
tress ; this has been shown in several laboratories 
utilizing in aggregate several hundred human 
subjects. (2) There is a linear relation between 
the intensity of anxiety, anger, or depression and 
plasma hydrocortisone levels. (3) Particularly 
high hydrocortisone levels occur in the presence of 
disintegrative anxiety. This finding has recently 
been followed and confirmed in an ingenious ex- 
periment by Dr. Sheldon Korchin and associates. 
Dr. Korchin joined the Adult Psychiatry Branch 
as visiting scientist during the year. (4) Re- 
markably consistent hydrocortisone elevations oc- 
cur in "first experience" situations — those char- 
acterized by a high degree of novelty and am- 
biguity. 

We have been pursuing some of these findings 
during the past year — partly by way of additional 
checking, partly searching out further implica- 
tions, partly inquiring whether epinephrine and 
norepinephrine are budged under these same 
circumstances. 

In current studies, we are attempting to relate 
fluctuations in emotional state to fluctuations in 
plasma and urinary levels of hydrocortisone, 
epinephrine, and norepinephrine. We are utiliz- 
ing three situations to observe changes in emo- 
tional state: (1) adaptation to new environment, 
(2) spontaneously occurring events in the life 
of each subject, (3) standard commercial films 
that effectively present common human problems. 
In each situation, modest but clearly detectable 
shifts in emotional state can be observed, and evi- 
dence is rapidly accumulating that there is con- 
comitant variation in levels of hydrocortisone, 
epinephrine, and norepinephrine. The fluctua- 
tions in emotional states are determined in several 
ways: (1) personal interviews, (2) ward observa- 
tions, (3) self-rating questionnaires, (4) projec- 
tive tests. In this work we have benefited from 
the collaboration of Dr. Joseph Handlon of the 
Laboratory of Psychology. 

Adaptation to a new environment has proved 
to be a potent stimulus for adrenal activity. We 
have found substantial hormone elevations in 
normal controls during the first few days follow- 
ing admission to the Clinical Center. This is a 
high-tension experience for most of these in- 
dividuals, since they typically have only a vague 
image of the experiences they are about to undergo 



and are concerned about possible risks. Under 
these circumstances we have observed not only 
significant elevations in plasma and urinary hy- 
drocortisone but also in the excretion of epineph- 
rine and norepinephrine. The norepinephrine 
elevations are more consistent than the epineph- 
rine elevations. This is in keeping with earlier 
findings suggesting that hydrocortisone and nor- 
epinephrine move closely together, while epineph- 
rine shows a somewhat different pattern of 
response to environmental stresses. 

Another point of interest has to do with differ- 
ences in the diurnal pattern of hydrocortisone 
levels during the first few days after admission. 
Almost all individuals show significant early 
morning elevations in comparison with their basal 
levels. However, some individuals maintain rela- 
tively high levels throughout the 24-hour period, 
while others show a sharp decline, sometimes hav- 
ing no detectable plasma steroids in afternoon and 
evening. The 24-hour urine, which is so ex- 
tensively used in medical research, only reflects 
these differences in diurnal patterns to a modest 
degree. Thus, if one were relying on 24-hour 
urines alone, a good deal of this difference in reg- 
ulatory pattern would be obscured. This may 
have methodological implications for other fields 
of medical research. At present, we are searching 
for possible behavioral correlates of the different 
neuroendocrine regulatory patterns. 

Our data this year have shown that individuals 
are quite consistent in their adrenocortical re- 
sponses. Some individuals consistently respond to 
the stresses of ordinary living with substantial 
steroid output; others show relatively slight re- 
sponse under the same circumstances. These 
differences tend to emerge more clearly under 
stress than under basal conditions. Thus, we ob- 
serve patterns of adrenocortical function that are 
characteristic of individuals, and we are trying to 
correlate these with enduring patterns of behavior 
that are also characteristic of the individual. In 
the near future we hope to put our working 
hypotheses on this problem to a predictive test. 

We are exploring various techniques for evok- 
ing emotional responses experimentally and meas- 
uring concomitant hormonal changes. Our main 
effort in this direction during 1959 has been the 
use of movies. In human stress research, it is es- 
sential to keep ethical considerations in mind when 



MENTAL HEALTH NEUROLOGICAL DISEASES AND BLINDNESS 



175 



formulating experimental plans for evoking re- 
liably observable emotional responses. Movies 
have the advantage of being clearly justifiable on 
ethical grounds. In addition, they permit gen- 
eralization to the common experiences of everyday 
living; they do not represent an extreme or un- 
usual psychological stress. 

Our findings with the movie technique so far 
may be briefly summarized as follows: (1) mod- 
erate prefilm hydrocortisone elevations occur con- 
sistently, just as pre-experimental elevations have 
occurred in other contexts ; these elevations are as- 
sociated with a background of uncertainty and 
tension about the experiment; (2) against the 
background of this tension and moderate steroid 
elevation, sharply contrasting film effects have 
been demonstrated : pleasant, absorbing films con- 
sistently produce a drop in steroid levels, whereas 
powerful films tend to produce further elevation. 
In connection with the latter films, an important 
methodological point is the "fit" of a given film 
with the attitudes of a particular audience. A 
film that constitutes a powerful stimulus for one 
type of audience may be a weak stimulus for a 
different sort of audience. 

Evidence has accumulated over the past few 
years that the pituitary-adrenal system is a sensi- 
tive reflector of the degree of behavioral arousal of 
the organism and presumably of tonic states of ex- 
citation in the CNS. If this is correct, we might 
expect to find evidence of such organismic arousal 
under conditions of elevated corticoid levels by 
studying what the brain does when it is not im- 
mediately interacting with the environment, as 
during sleep. By seeking such relationship we 
hope to further elucidate the psychophysiological 
adaptation of the organisms to stress, to explore 
individual differences in reaction to stress, and 
possibly to further understanding of those mal- 
adaptations to stress which contribute to mental 
and psychosomatic illness. Thus, we are seeking 
a possible relationship between adrenocortical 
function and the quality of sleep and dreaming, 
employing the latter as behavioral indices of tonic 
states of organismic arousal. 

Electroencephalographic and electroculographic 
records are obtained of sleeping subjects, concur- 
rently with the study of their blood and urine 17- 
hydroxycorticosterone levels. Both types of 
procedures are carried out under circumstances 



when the corticoid levels are elevated, and when 
they are at stable baseline levels for the subjects 
under study. Under these contrasting conditions, 
the electrophysiological measures are assessed in 
terms of sleep depth and the number and duration 
of dream periods, while the manifest dream con- 
tent is rated on a dimension of "threat" expres- 
sion. Thus far, the subjects have been normal 
volunteer controls. Data collection under the con- 
ditions described has now begun. The first phase 
of the study has been concerned with studying 
techniques of assessing the depth of sleep, the oc- 
currence of dreaming, and the collection and rat- 
ing of dream content. 

In our Personality Development Section, we 
have selected for study one of the commonly oc- 
curring experiences which may stimulate personal 
growth for many people, but may also lead to 
breakdown for others: the transition from high 
school to college. Our aim is to determine some of 
the sources of problem-solving effectiveness in 
adolescence. We hope to learn about the kinds of 
factors in our subjects' life experience, family, per- 
sonality, and current environment which seem to 
be related to their finding effective ways of dealing 
with stress during the period under study. 

Under the leadership of Dr. Earle Silber, we se- 
lected a group of students on the basis of the fol- 
lowing minimal criteria of mental health: (1) 
evidence of competence in school work, (2) evi- 
dence of ability to make and maintain interper- 
sonal closeness with at least one person outside the 
immediate family. All the students in our sample 
were exposed to the demands of a college prepara- 
tory course in a good public high school ; most of 
them had a similar socio-economic background. 
The sample was drawn from the senior class at the 
Bethesda-Chevy Chase High School, which num- 
bered 530 students, about 53 percent of whom were 
girls. There are 20 students in the sample we 
chose for intensive study. 

In selecting the 20 students the following steps 
were carried out : all of the students were in the 
senior class, were exposed to a short talk telling 
about the project and asking for their cooperation. 
Then a letter was sent to all senior-class students, 
and the students were asked to sign this letter if 
they wished to volunteer. Parental signatures 
were also required. The letters were then returned 
to the school, and also gave consent for us to have 



176 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



copies of the students' transcript records. We 
picked students in the top half of the class who 
would be going to colleges not too distant, so that 
we could visit them if we desired, and also students 
who seemed to reveal some concentrated interest 
as reflected in their extra-curricular activities; 
and also those who seemed to have made a favor- 
able impression upon their teachers as evidenced 
by personality ratings, which was part of the tran- 
script record. From a volunteer group of about 
100, we selected 39 as probably meeting our crite- 
ria, and carried out a screening interview with 
them. The 20 intensive cases were chosen from 
these 39 and were seen for a series of weekly 
interviews. 

A special projective test was constructed by Dr. 
George Coelho and Dr. Silber, patterned after the 
Thematic Apperception Test, consisting of 11 
pictures designed to elicit characteristic modes of 
dealing with certain potentially stressful situa- 
tions. There were two sets of pictures — one for 
boys and one for girls — including scenes such 
as posting of final grades and couples sitting close 
in a car. 

Content of the subsequent interviews included 
investigation of the following areas: academic 
work, peer group relations, values, constructive ex- 
periences during high school, dating, plans for 
marriage, the family situation, areas of responsi- 
bility within the family, description of the par- 
ents, decisions about going to college, anticipation 
of college life, plans for living at college, 
academic preparation, and attitudes about college 
organizations. 

During the summer months we continued our 
interviewing to obtain information about the 
student's general history, medical history, and 
personal development, as well as to keep in touch 
with some of the events in his current experience. 
To understand more of the dominant values of the 
high school and its formal and informal organiza- 
tions, and the background against which our in- 
dividual subjects operated (that is, the current 
high school environment) , a series of four group 
interviews — along with the individual inter- 
views — was held with four students designated as 
group leaders. 

We have also gotten acquainted with the par- 
ents of our students and during the summer 
months, all of the parents (with a few excep- 



tions) were seen in a joint interview. We plan to 
continue interviews with the parents. We have, 
wherever possible, seen our students just before 
departure for college, to see what changes may be 
present on the eve of actually leaving home. 

Essentially then, we have had an opportunity 
to get acquainted with a group of 20 college- 
bound students while they were still in high 
school. By beginning with each student while he 
was still in high school, we had an unusual oppor- 
tunity to study the techniques of personal prob- 
lem-solving in the high school years, as well as the 
anticipatory phase of the transition to college. 
We have also had an opportunity to get ac- 
quainted with the student's past history and to 
have some direct experience with his parents. We 
have now begun interviewing our students after 
they have moved into the freshman year at col- 
lege. We made a field visit to each of the col- 
leges to see the student after he had been away 
at college 6 to 8 weeks. We interviewed some of 
the students when they were home for Thanks- 
giving, all of them during Christmas vacation, 
and plan to see each one again during Easter 
vacation. At the end of the freshman year, we 
plan another intensive series of interviews to re- 
view the entire year. This field study has bene- 
fited from our collaboration with Drs. Morris 
Rosenberg and Leonard Pearlin of the Labora- 
tory of Socio-Environmental Studies. 

Our interest has been in clarifying the behavior 
of competent adolescents, and particularly in 
learning how they cope with the stresses of this 
transitional period. Further, we are attempting 
to make systematic comparisons of these students 
with those who break down during the freshman 
year and are hospitalized in the Clinical Center. 

In order to study sources of interference and 
conflict in the developmental process, we have hos- 
pitalized a group of adolescents in whom there 
has been a significant area of developmental fail- 
ure, serious enough to make adaptation to the 
demands of a first-year college experience tem- 
porarily impossible. We selected the situation of 
the freshman in college because it provided an op- 
portunity to observe the impact of a new situation 
with new demands, both internal and environ- 
mental, upon the adolescent. 

The 3-East project has been in operation since 
January 1959, under the leadership of Dr. Roger 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



177 



Shapiro and Dr. Harold Greenberg. Since the 
opening of the ward we have hospitalized twelve 
college freshmen who were in sufficient emotional 
difficulty to make it advisable that they leave 
school. These patients have represented a wide 
range of diagnostic categories including acute 
schizophrenic reaction, paranoid personality dis- 
order, other types of character disorders, and some 
symptom neuroses. 

Our first efforts with this group involved organ- 
izing an effective program of therapy. This has 
included individual psychotherapy on a 3 or 4 
hour a week schedule for all patients, meetings of 
the entire patient group twice a week, the develop- 
ment of an activities program, and therapeutic in- 
terviews with the patients' families. 

Some of the research questions we hope to 
answer from data available from the therapeutic 
program. Other questions we are trying to get at 
in specially designed research interviews with the 
patients, their families, and their friends. Our 
questions up to now have been essentially in two 
areas. The first area includes questions about the 
nature of the new and presumably stressful situa- 
tion (the experience of the college freshman) , and 
about the psychological equipment the individual 
brings into the situation ; what his resources are in 
coping with it, what directions his adaptive efforts 
take. The second area includes questions about 
factors that might determine such behavior in in- 
dividuals, and have up to now been chiefly in- 
vestigations into aspects of his previous life 
experience which might account for some of the 
idiosyncratic responses we observe. In addition 
to information along these lines available from the 
data of individual therapy, we are now in process 
of designing a standardized interview in which 
one of us will attempt to get at this material 
specifically with each patient. 

Several other sources of data about the patients' 
adaptive efforts in the college situation are avail- 
able. One is the observations of friends, teachers, 
counsellors, etc., close to the patient during his at- 
tempts to adjust in his first year of college. We 
have sent one of our staff to the college setting in 
which each patient has experienced difficulty, to 
interview a number of people who were close to the 
patient, and get a picture from them of the coping 
efforts made by the patient and the kinds of situa- 
tions he had to cope with there. We have been 



particularly interested in the kinds of efforts the 
patient made to form friendships in the college 
setting and have addressed many questions to the 
patients' friends about the nature and quality of 
relationship that existed between them and the 
patient. 

We have attempted to use observation of the pa- 
tient's adaptation to the new experience of hos- 
pitalization and living in the ward situation as 
another source of data about the nature and qual- 
ity of his coping efforts. Again we have been par- 
ticularly interested in how he goes about forming 
new relationships and have asked nursing per- 
sonnel to make an ongoing record of the develop- 
ing relationship between themselves and the pa- 
tient. We hope to learn how these patients relate 
to a variety of individuals, and if we can to clarify 
why for them formation of new relationships as a 
type of adaptive behavior is so difficult and when 
it occurs is often unsuccessful in reducing their 
anxiety and maintaining their self esteem. 

With regard to the second area of questions, 
most of our efforts to understand the personality 
development of our patients have involved study 
of relationships within the families in which they 
have grown up. Interviews with parents and 
siblings have been conducted with particular in- 
terest in what could be learned about their con- 
scious and unconscious attitudes toward the pa- 
tient, their feelings about the patient's assets, their 
image of his liabilities, and their readiness to see 
him as a separate individual. We also want to ob- 
tain from the parents as thorough a picture as 
possible of their own experience as adolescents. 
As they recall their own adolescence, we hope to 
gain some knowledge of what sources of anxiety 
existed for them as adolescents and how they dealt 
with them. We then want to determine how 
much their child's adolescence has revived this 
anxiety in them, and how much they have re- 
sponded with anxiety to their child's explorations 
in an area of anxiety in his own adolescence. This 
may tell us something about the developmental ex- 
periences which promote and which impair adap- 
tive behavior in adolescence. 

During 1959 the Section on Family Studies, 
under the leadership of Dr. Lyman Wynne, has 
deepened its investigations of family relations in 
schizophrenia. Broadly speaking, the work of 
the past year has strengthened previous impres- 



178 



A]SnSTUAL REVIEW OF INTRAMURAL RESEARCH 



sions that the familial patterns of interpersonal 
relationship of schizophrenic patients differ from 
those of nonschizophrenic psychiatric patients 
and that the schizophrenic offspring occupies a 
different family role and has had a different famil- 
ial experience from nonschizophrenic siblings. 

Two independent approaches have been useful 
in producing new ideas and data which bear upon 
these problems: family psychotherapy, and psy- 
chological testing of entire families. All 25 fami- 
lies which have been studied intensively in this 
project during the past 2 years have been seen 
in "family therapy" in which both parents, hos- 
pitalized patient, and siblings are seen together 
in exploratory, psychoanalytically oriented, psy- 
chotherapy. In effect, the communication and 
interpersonal patterns of the family system are 
not reconstructed in family therapy but are ob- 
served directly. The very recent addition to the 
facilities of one-way observation windows to the 
interview rooms has expanded the research op- 
portunities in studying the family therapy 
sessions. 

The family-therapy approach has led to the 
conclusion that the traditional use of individual 
interview descriptions of family interaction is of 
very dubious reliability. Repeatedly, when family 
members describe disturbing events that have just 
happened within a therapy session, major aspects 
of the events are distorted or omitted by all the 
family members. However, direct observation il- 
luminates behavior that could not be reported by 
the family members because it has occurred out- 
side their awareness. 

Although this project is primarily oriented 
toward untangling the relation of family patterns 
and schizophrenia, the use of family therapy as 
an exploratory research tool has led to a significant 
byproduct: a contribution to the development of 
the theory and technique of family therapy, an 
approach which has recently achieved considerable 
recognition as a new and potentially valuable 
addition to the therapeutic repertory of psychia- 
trists. Observations and ideas within our group 
about the advantages and limitations of family 
therapy, including the relation of family therapy 
to concomitant individual psychotherapy, have 
been evolving into a conceptual framework during 
the past year. 

The criteria for selection of families compared 



in this project have minimized all presenting dif- 
ferences except one: the presence of a schizo- 
phrenic (or psychiatrically ill and hospitalized), 
versus a nonschizophrenic young adult offspring. 
In other respects, the families can all be character- 
ized as consisting of two parents and at least one 
sibling of the presenting patient, as having pre- 
dominantly American middle-class customs and 
values, and as sharing a willingness to explore 
jointly the difficulties involved in the psychiatric 
illness. 

During the past year procedures for evaluating 
both the diagnostic and the sociocultural back- 
ground characteristics of the families selected have 
been systematized and sharpened. Beginning 
with background interviews by the psychiatric 
social workers of the project, a procedure for re- 
cording historical material as it cumulatively un- 
folds has been developed. 

In comparing the families of schizophrenic and 
of nonschizophrenic psychiatric patients, the work 
in family therapy has been useful in suggesting a 
number of hypotheses of significant differences. 
For example, the familial response to "deviant" 
behavior of a schizophrenic family member (that 
is, behavior outside the code or subculture of the 
particular family) generally has the effect of frag- 
menting the behavior into concrete aspects; for 
example, by ignoring the most obvious intentions 
of the deviant family member and giving only a 
very limited, literal interpretation to a trivial 
aspect of the behavior, such family responses re- 
duce and confuse the central meaning and intent 
of the behavior into incoherence and uncertainty 
about the validity and authenticity of one's own 
perceptions and intentions. In contrast, the 
familial response to "deviant" behavior in non- 
schizophrenic families typically involves, for ex- 
ample, relatively straightforward accusations and 
the production of guilt in the deviant person, but 
without reducing his capacity to trust his own 
senses about what he intended. 

Such hypotheses, as they become progressively 
refined, seem to lend themselves to systematic 
comparison studies relevant to basic features of 
schizophrenic experience. Procedures are now 
being developed for making systematic use of 
family therapy transcripts in such comparison 
studies and plans are under way to compare these 
and other families, using standardized procedures 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



179 



focused on particular aspects of these hypotheses. 

In addition to family and individual inter- 
views, three other sources of data have expanded 
in their contribution to the family studies pro- 
gram during the past year: (a) nursing observa- 
tions, both the traditional observation of 
individual patients and the observation of pa- 
tients interacting with their families, who are en- 
couraged to visit on the nursing unit; (b) home 
visits, with the entire family present, have be- 
come a regular part of the work with all families ; 
naturalistic observations have been made for 
various lengths of stay by staff members having 
different roles — nurse, social worker, psychiatrist; 
and (c) art therapy, as a tool for diagnostic and 
therapeutic work with entire families, as well as 
individuals, has, in selected instances, been quite 
informative. 

Parallel to the direct clinical work with fam- 
ilies, a consultant to the project, Dr. Margaret 
Thaler Singer, has been studying "blind" the 
psychological test protocols obtained from entire 
families seen in the project. Given first the pro- 
tocols of the family members but not of the pa- 
tient, Dr. Singer predicts which families have a 
schizophrenic member and then, given the patient 
protocols, matches patient with family. Thus 
far, with eleven families, a remarkable accuracy 
has been achieved. Most interestingly, these re- 
sults could not be obtained using traditional 
methods of interpreting test protocols by their 
content. However, when attention was paid to 
the form of the thinking used by the subjects — 
for example, in the way in which attention and 
meaning were manipulated — then accuracy of 
differentiation was possible. The convergence of 
these test findings with the clinical findings pre- 
viously mentioned has been striking and will be 
intensively examined in further evaluations of the 
material already obtained and in future investiga- 
tions of additional families. 



Clinical Neuropharmacology Research 

Center* 

The past year has seen an increasingly fruitful 
interaction between the CNRC and Saint Eliza- 
beths Hospital, and has further defined areas of 



♦Prepared by the Chief, Joel Elkes, M.D. 
556044—60 13 



mutually complementary interest. This interac- 
tion has been manifest in a number of steps taken 
by the Hospital to assist the program during its 
formative period. Dr. N. Waldrop, formerly 
Chief of the William A. White Service, has been 
promoted to the newly created post of Associate 
Director of Research for Saint Elizabeths Hos- 
pital, and, in this capacity, has given valuable 
assistance in the implementation of the CNRC 
program within the total Hospital setting. A 
new Biometrics Branch has been created by the 
Hospital. This is to collaborate with appropriate 
personnel of the CNRC, and the Biometrics 
Branch of NIMH, in meeting the statistical needs 
of the program. The Chief of CNRC, and the 
Director of Training of the Hospital are actively 
exploring ways and means whereby the training 
potential of the CNRC could be used to optimum 
advantage, without unduly infringing on the time 
of CNRC personnel. Already, requests have been 
received from residents of the Hospital to work in 
the laboratories of CNRC over and above their 
normal duties, and a resident has been assigned 
to the CNRC by the Hospital to collaborate in an 
ongoing program of clinical investigation in the 
wards of the William A. White Service. The 
regular internal and guest seminars of CNRC 
have attracted wide participation from the Hos- 
pital. Taken together with joint staff confer- 
ences, regular teaching rounds within the Wil- 
liam A. White Building, and the spontaneous 
formation of small groups centered around sub- 
jects of common interest, they have greatly 
strengthened the links (both personal and ad- 
ministrative) between CNRC and the Hospital. 
The Research Committee of the Hospital, of 
which the Chief, CNRC, is Chairman, now com- 
prises four members of the CNRC staff (Dr. I. 
Whitfield, Dr. H. Weil-Malherbe, Dr. G. C. 
Salmoiraghi, and Dr. M. Hamilton). The work 
of this Committee (which meets at monthly inter- 
vals to consider research proposals, both from the 
CNRC and the Hospital) has gone some way 
towards defining standards, and devising proce- 
dures designed to further research within Saint 
Elizabeths Hospital. 

Despite these hopeful trends, however, it would 
be idle to ignore some deep-seated difficulties which 
confront the CNRC and the Hospital in the pur- 
suit of an advanced program of research and of 



180 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



training. Staff shortage, at professional and non- 
professional levels, takes first place, and is acutely 
felt in the William A. White Service, as well as 
in other parts of the Hospital. For similar 
reasons (and despite every effort to participate in 
a common research program) the Pathological 
Laboratory of the Hospital can only meet a very 
limited number of requests for studies arising out 
of the program; a priority system had to be 
strictly adhered to in the allocation of research 
projects requiring such laboratory studies. 
Furthermore, a number of medico-legal issues con- 
nected with the pursuit of clinical investigations 
within the confines of the Hospital remain un- 
resolved, thus favoring the use of established 
forms of treatment, and holding in abeyance the 
introduction of procedures of a more novel and 
exploratory nature. Lastly, the lack of intermedi- 
ate treatment facilities such as Out-Patient, Day 
Hospital, Night Hospital, and Club facilities is 
being increasingly felt by the CNRC, as the re- 
habilitation of the Center, and the Hospital, 
gathers momentum. The Hospital is fully aware 
of these needs, and, within the stringent financial 
limitations imposed upon it by present circum- 
stances, is doing its best to bring these facilities 
into being. 

There is one further aspect which has become 
clearly apparent during the past year of the 
Center's operation. The physical separation of 
CNRC from the Clinical Center at Bethesda 
has been increasingly felt by members of staff, 
and the hope has been steadily expressed that some 
aspects of the neuropharmacology program could, 
with advantage, be pursued at the Clinical Center. 
The physical representation of the subject of 
neuropharmacology, both at Saint Elizabeths Hos- 
pital and at the Clinical Center, would make 
for more ready interaction between the program 
of CNRC and related aspects of the programs of 
the Laboratories of Clinical Science, Adult Psy- 
chiatry, and Psychology of Clinical Investiga- 
tions, NIMH. Furthermore, it would also 
encourage the circulation of personnel between the 
Clinical Center and the CNRC facility, and thus 
reduce the sense of isolation of CNRC personnel, 
as well as increase the contact between Saint Eliza- 
beths Hospital and the Clinical Center. There is, 
in fact, every evidence that such a move would 
greatly enhance the mental climate within both 



the CNRC and the Saint Elizabeths campus, and, 
through closer liaison of the Hospital with the 
Clinical Center, contribute to the steady evolution 
of an academic setting and attitude within the 
Hospital. 

For the purposes of the present report, the divi- 
stion of the Center into Sections of Psychiatry, 
Chemical Pharmacology, and Behavioral Sciences 
is adhered to. It has, however, become increas- 
ingly apparent that, while serving a very useful 
purpose during the early formative period of the 
Center, these terms no longer fully connote the 
principal themes to which the program of CNRC 
wishes to be committed. It is hoped that these 
themes may receive their formal recognition in an 
internal reorganization of the Center during the 
coming year. During the interim, Dr. G. C. Sal- 
moiraghi has made a valuable contribution to the 
evolution of the Center by assuming responsibility 
for central laboratory services common to all Sec- 
tions, by assisting the Chief, CNRC, in the co- 
ordination of the programs as a whole, and by 
representing him on appropriate occasions. 

SECTION ON PSYCHIATRY 

The slender resources of the William A. White 
Service of Saint Elizabeths Hospital have en- 
couraged Drs. A. Hordern, of CNRC, and J. 
Lofft, of Saint Elizabeths Hospital, to initiate a 
comparative study of two phenothiazines within 
six wards of the Service. The broad aims of this 
study were essentially fourfold. In the first place, 
it was hoped to explore the limits of feasibility 
of a number of procedures, and to determine their 
most economical use. Secondly, it was hoped to 
define ways and means whereby the mere pursuit 
of such a study could contribute maximally to the 
training of staff, and thus to the care of patients 
not directly involved in the study. A third aim 
was to test various clinical research instruments 
(and more particularly rating scales) in terms of 
their usefulness and reliability in the day-to-day 
setting of a busy mental hospital ward. Finally, 
it was hoped to determine the effect of such a clini- 
cal research program on the attitudes and aspira- 
tions of the staff. 

In this preparatory study, therefore, informa- 
tion regarding the relative merits of the two drugs 
used (Trifluoperazine and Prochlorperazine) was 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



181 



regarded as incidental; the real yield lay in a 
contribution to the solution of some controversial 
methodological problems, of which seven received 
particular emphasis. These were (1) the role of 
structural milieu in ward management; (2) the 
effect of interpersonal milieu, and nursing care in 
the management of patients; (3) the mobilization 
and cultivation of latent research skills in the 
nursing staff; (4) the design of the type of trial 
best suited to meet the requirements of (1), (2), 
(3) ; (5) the assessment of patient behavior; (6) 
the assessment of attitudes towards a clinical re- 
search program in a hospital ward; and, finally 
(7) the most economical way of collecting, tabu- 
lating, and processing data. Although the study 
involved only 24 specially selected patients, the 
simultaneous pursuit of the program in 6 wards 
proved an interesting, and (from the point of view 
of staff training) helpful feature. The patients 
were divided into six groups of four, and were ob- 
served in special day rooms (decorated by the pa- 
tients) in six wards of the Service. In each ward 
the group of four formed the nucleus of a larger 
group of ten patients, the six additional members 
consisting of chronic schizophrenics receiving 
their customary medication. The six groups were 
consistently nursed by selected day and evening 
nursing assistants, a carefully defined routine be- 
ing followed in each case. Regular group discus- 
sions with personnel contributed to the training of 
personnel, and familiarized them with the regular 
instruments being used. Thirteen different rating 
scales, the details of which are given elsewhere, 
were employed. According to needs these were 
administered at daily, twice weekly, weekly, 
monthly, and three-monthly intervals. 

The results of this study to date are distinctly 
encouraging. The methods have proved feasible, 
and economical of staff time. Personnel attitudes 
throughout the study have been monitored. A 
trial of this kind would appear to be an economical 
training device in group nursing and rehabilita- 
tion techniques. There appears little question of 
its positive effect on the mental climate of the 
various wards. At the initiative of the staff of 
Saint Elizabeths Hospital, the study is now to be 
extended to include a further 126 patients who are 
to be transferred especially to the William A. 
White Service for rehabilitation ; it is hoped that 
parallel studies will be initiated in other services 



of the Hospital. A visual aid exhibit, prepared 
by the principal investigators, has been a useful 
demonstration and teaching device, and is to be 
shown in an expanded form at a forthcoming meet- 
ing of the American Psychiatric Association. 

In an attempt to study social interaction within 
a chronic mental hospital ward in a quantitative 
way, Dr. S. Kellam is developing an objective 
method for the visual recording, and measure- 
ment of the relative association, or isolation, of the 
individual patients within the ward setting. In- 
formation regarding the number of contacts of a 
patient is noted by nurses and aides on a time- 
sample basis, and recorded on a specially con- 
structed grid by means of mobile map tacks. The 
clustering of these tacks readily reflects -social 
contacts, a ward log supplementing this visual de- 
vice. The method is already giving useful objec- 
tive indications of the group structure of the ward, 
and the prestige systems existing in the wards. It 
should be of particular interest in an examination 
of devices or procedures (including drugs) de- 
signed to render the patient more accessible to the 
milieu in which he functions. 

In an attempt to study factors making for 
chronic hospitalization, Dr. D. Lipsitt has ex- 
amined the role of so-called "dependency" in the 
psychiatric in-patient, and of the possible, though 
unintended, collusion between the needs of the 
over-dependent patient and an over-protective at- 
titude in a ward milieu. The term "dependency" 
is in obvious need of clarification and measure- 
ment, Two hundred patients form the subject of 
this study. The instruments used include selected 
scales from the Minnesota Multiphasic Person- 
ality Inventory, a review and scoring of the pa- 
tient's case record, a psychiatric interview, and 
behavior ratings ; other instruments are being de- 
veloped. Present trends indicate that long-term 
patients deny their dependency (showing a 
higher denial and a low dependency score), 
whereas patients admitted for the second time 
tend to accept, admit, and overtly display their 
need for dependency. It is hoped that, with 
growing experience in this area, dependency scores 
may be tested for predictive value and, perhaps, 
be put to use in selecting appropriate treatments 
for patients with varying dependency needs. 

In a cognate vein, Dr. M. Geller is developing 
methods for study of the transition of the chronic 



182 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



schizophrenic patient into the community, with 
special reference to the use of the pharmacothera- 
pies and group therapeutic techniques to facilitate 
such transition. Two matched groups of eight 
patients are being used as a pilot sample in this 
study. Experience in the conduct of group ses- 
sions with chronic schizophrenics and in the re- 
cording and analysis of data is being gained at 
present, though this, of necessity, is a slow process. 
It is hoped that utilization of analytically oriented 
group psychotherapy may, in time, allow one to 
exploit the assets of the chronic patients during 
trial visits and during an initial period (of, say, 
6 months) following discharge. It is also hoped 
that by providing regular group therapy during 
this stage of transition the patient may be success- 
fully weaned from his undue dependence on the 
hospital and encouraged to make a successful ad- 
justment in the community. 

Drs. Harwood, Hordern, and Lofft, together 
with a number of other colleagues, are studying 
the effects of Imipramine on plasma 17-hydroxy- 
corticoid levels in eight depressive patients, and 
are attempting to correlate these findings with 
clinical responses to the drug, as well as its effect 
on protein-bound iodine and plasma catecholamine 
levels. This study is in progress ; no biochemical 
data is as yet at hand. 

Dr. R. Gentry, CNRC, and Dr. N. Waldrop, of 
Saint Elizabeths Hospital, are gaining experience 
in the use of an optical transducer device for the 
study of changes in finger blood flow. This is to 
be used in a number of psychophysiological in- 
vestigations, and may be found particularly help- 
ful as an index of autonomic activity in word- 
association studies. 

SECTION ON CHEMICAL PHARMACOLOGY 

In continuation of previous work, Dr. Weil- 
Malherbe has attempted to refine his published 
method for estimating epinephrine and norepi- 
nephrine in plasma. It was found that the results 
obtained by the trihydroxyindole method were 
only slightly lower than those obtained by the 
ethylene diamine method. Studies on the chemical 
mechanisms of ethylene diamine condensation in- 
dicate that in the reaction with epinephrine a sin- 
gle compound is formed as a major product and 
that side reactions occur to an insignificant degree. 



In the reaction with norepinephrine, on the other 
hand, two or three major products are formed 
from catechol and dihydroxy-mandelic acid. The 
need for a reliable method for estimating cate- 
cholamines is an urgent and persistent problem, 
and it is hoped that Dr. Weil-Malherbe's critical 
reappraisal of existing methods (including his 
own) may be a useful contribution to the field. 

In a cognate area, Dr. H. Weil-Malherbe and 
Dr. E. R. B. Smith are attempting to develop a 
method for estimating urinary metanephrine and 
normetanephrine by a modification of the method 
of Euler and Floding. The zinc ion was found to 
be an essential catalyst in the formation of fluores- 
cent derivatives of these two compounds. Taken 
together with the methods now being developed 
in the Laboratory of Clinical Science, Clinical 
Investigations, NIMH, the availability of a quan- 
titative method for the estimation of metanephrine 
and normetanephrine in urine should make it 
possible to account for the major metabolites of 
epinephrine and norepinephrine in man. It is 
intended to put these methods to use in appro- 
priate pharmacological studies. 

Dr. H. Weil-Malherbe and Dr. H. Posner are 
examining the effect of DOPA on the synthesis of 
catecholamines in the brain after depletion by 
reserpine. It had been shown in previous work 
that the intravenous injection of DOPA accel- 
erated the reappearance of catecholamines in rab- 
bit brain and, equally, that there was a significant 
redistribution of intracellular epinephrine, nore- 
pinephrine, and hydroxytryamine following ad- 
ministration of reserpine. The effect of Dopamine 
on these processes is being examined further. 

In pursuit of previous studies, Dr. Szara is now 
developing quantitative methods for the estima- 
tion of the ^-hydroxy derivatives of psycho- 
tomimetic tryptamine derivatives, with special 
reference to the estimation 6-hydroydimeth- 
yltryptamine and (J-hydroxydiethyltryptamine. 
This has involved the organic synthesis of refer- 
ence materials, and a comparison of these with 
material isolated from urine. A sensitive and 
specific spectrophotometric method, measuring 
1/jttg/ml of tfOhydroxydiethyltryptamine in urine 
had been developed. This has been found useful 
in following the £-hydroxylation pathway of di- 
ethyltryptamine, a compound shown by Dr. Szara 
to possess marked psychotomimetic properties. 



MENTAL HEALTH— NEUROLOGICAL DISEASES AND BLINDNESS 



183 



In a promising juxtaposition of biochemical 
method with animal behavioral techniques, Dr. 
Szara, of this Section, and Dr. Eliot Hearst, of the 
Section of Behavioral Sciences, have attempted 
to study the relation of the rate of tfOhydroxyla- 
tion of diethyltryptamine to the threshold re- 
quired to elicit behavioral change in the individual 
rat. It "was found that the rate of transforma- 
tion of dimethyltryptamine to its ^-hydroxy deri- 
vative differed from animal to animal, and that 
the rate correlated well with the thresholds needed 
to elicit such behavioral effects. The evidence 
thus pointed to £-hydroxylation as an important, 
and possibly essential, step in the production of 
behavioral change by tryptamine derivatives. It 
is also of interest that the £-hydroxylation of di- 
ethyltryptamine by a microsomal enzyme system 
provides one of the few instances of the produc- 
tion of a psychoactive agent in vivo. It will be 
of interest to examine the effects of ataratic and 
other agents on the rate of this tf-hydroxylation 
process. 

Dr. H. Posner has been following the metabo- 
lism of some phenothiazines in man, with special 
reference to possible excretion of phenolic 
metabolites in urine. By incubation of urine ex- 
tracts with glucuronidase, he has obtained evidence 
suggesting that giucuronides constitute a con- 
siderably larger fraction of the excreted metab- 
olites than sulfoxide derivatives. In an ad- 
mittedly difficult study, Dr. Posner is attempting 
to develop methods of identification and analysis 
of the various metabolites of chlorpromazine and 
is hoping to use these in an intensive study, both 
of excretion patterns of phenothiazines in the in- 
dividual patient, and in studies of the behavioral 
effects of a number of key metabolites in the ex- 
perimental animal. As in the case of Dr. Szara, 
Dr. Posner is planning his biochemical studies in 
conjunction with Dr. Eliot Hearst of the Section 
of Behavioral Sciences ; both studies are attempts 
to correlate individual biochemical variation in 
the metabolic handling of a drug with behavioral 
effects and thresholds for such behavioral effects. 

Dr. Posner has also carried out an extensive 
survey of the urines of selected patients with the 
view to isolating suitable case material (such as 
phenylketonuria, alcaptonuria, tyrosinosis, pro- 
phyrinuria) for subsequent study. Although this 
survey of some 286 patients did not yield the 



material intended, it drew attention to certain 
anomalies in the Ehrlic indole reaction, which led 
Dr. Posner to examine the possible role of metabo- 
lites of chlorpromazine in this reaction. It is this 
incidental finding which led Dr. Posner to his 
present study of the disposition of the pheno- 
thiazine derivatives in man. 

In conjunction with Dr. A. H. Stewart of Saint 
Elizabeths Hospital, Dr. T. Harwood has studied 
the effects on plasma 17-hydroxycorticoid levels in 
six patients being treated with the drug for symp- 
toms of depression. In keeping with previous 
findings by others, the 17-hydroxycorticoid plasma 
levels were found elevated in these patients prior 
to treatment. There was a fall in these levels fol- 
lowing iproniazid administration ; though the de- 
gree of clinical response did not necessarily cor- 
relate with this alteration in 17-hydroxycorticoid 
concentration. 

Dr. Cambosos has been engaged in the recalibra- 
tion and development of the Chaney colorimetric 
procedure for the determination of protein bound 
iodine levels in serum. The modification has in- 
creased the sensitivity of the method about five- 
fold, making possible the determination of PBI 
levels in about 0.1 ml. of serum. This should thus 
prove useful in longitudinal studies (both clinical 
and experimental) where frequent blood sampling 
may be required. 

SECTION ON BEHAVIORAL SCIENCES 

The Section of Behavioral Sciences has con- 
tinued its studies of the mechanisms subserving 
the coding of information along the auditory 
pathway, with special reference to an analysis of 
the role of inhibitory mechanisms in the cochlear 
nucleus. One such study, carried out by Miss P. 
Stopp, has centered on an analysis of unit response 
patterns of the avian auditory pathway, as fol- 
lowed by microelectrode, within the nucleus mag- 
nocellularis of the pigeon. It was found that, 
despite a lack of differentiation of the cochlear 
receptors in birds into inner and outer hair cells, 
the pattern obtained showed remarkable similarity 
to those observed in the much more highly or- 
ganized auditory system of the cat. 

In another study Dr. J. B. Fotheringham has 
attempted to influence the inhibition of unit activ- 
ity normally seen in the inferior colliculus of the 
cat following appropriate two-tone stimulation. 



184 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



A number of well-known centrally acting drugs 
were used. Significantly, none of these noticeably 
affected the discrete inhibitory process operating 
at this level. A similar lack of responsiveness was 
noted in the cochlear nucleus by Dr. A. S. 
Schwartz. The results thus tentatively suggest 
the existence of local (and possibly pericellular) 
barriers at high central level. In an attempt to 
test for these, pericellular microinjection will be 
attempted, though of necessity the experimental 
hazards of this procedure are considerable. 

Dr. E. Gumnit has continued to study direct 
current changes in the auditory cortex of the cat in 
response to auditory stimulation. In view of 
the sensitivity of these changes to slight variations 
in experimental condition (such as degree of 
anaesthesia, moisture, and temperature of the 
cortex) rigorous control of these had to be assured, 
and optimum conditions defined. A cell suitable 
for direct current recording has been devised and 
tested. Results to date suggest the existence of 
direct current changes in the auditory cortex, and 
particularly a localization of these changes to 
Auditory Area I. The role of direct current 
changes in the handling of sensory information is 
still controversial, and any established facts in this 
area must be judged welcome. 

Dr. E. P. Michael, a Guest Worker at the Center 
on a Eockef eller fellowship, has made a useful be- 
ginning in developing a cannulation and micro- 
infusion technique, which, it is hoped, may make 
it possible to slowly inject small amounts of drugs 
directly into selected areas of the brain in the con- 
scious monkey subjected to simultaneous behav- 
ioral study in an operant conditioning situation. 
So far, the method has been used in four Ehesus 
monkeys; approximate thresholds for chlorpro- 
mazine, injected directly into areas of the reticular 
formation, have been obtained. It is quite obvious, 
however, that a refinement of the technique is es- 
sential before any further study in this area can be 
undertaken. Time expended in developing this 
technique, however, may be well worth while, since 
(particularly if combined with biochemical and 
endocrinological studies) it may offer a reasonably 
direct approach to an analysis of the regional 
physiology and pharmacology of the brain stem. 
It is hoped that following Dr. Michael's return to 
England the work may be continued by other mem- 
bers of the group. 



An independent area of investigation is being 
opened up at the Center by Dr. G. C. Salmoiraghi. 
In previous work, carried out with D. B. Burns, at 
McGill University, Montreal, Canada, Dr. Salmoi- 
raghi, using extracellular microelectrodes, had at- 
tempted to determine the location and pattern of 
discharge of respiratory neurones in the brain 
stem, and to account for the rhythmic nature of 
respiration in terms of reciprocating inhibitory 
mechanisms. He is now hoping to extend these 
studies, and, more particularly, to elucidate fur- 
ther the mode of action of C0 2 and of other 
drugs on these mechanisms. The technical 
difficulties (particularly those connected with 
movement artefact) are very considerable; yet it 
is hoped that an elucidation of the mode of action 
of drugs on rhythmically discharging cells of the 
medulla may contribute to an understanding of the 
action of drugs on rhythmic processes elsewhere 
in the brain stem. Furthermore, the concepts 
developed by Dr. Salmoiraghi to account for the 
discharge of respiratory neurones may find their 
application in the medullary centers controlling 
cardiovascular phenomena. In view of the strik- 
ing involvement of central autonomic centers in 
some phases of stress, and of mental disorder and 
the effect of drugs on such phenomena, a discrete 
analysis of the organization of these central steer- 
ing mechanisms is deemed desirable, despite the 
long-term nature of such an undertaking. 

Dr. Eliot Hearst, in conjunction with Dr. Mur- 
ray Sidman of the Department of Experimental 
Psychology, "Walter Eeed Army Institute of Ee- 
search, has continued a study on the aversive na- 
ture of a conflict producing stimulus in the rat. 
Animals were trained in situations which produced 
conflict, but which, equally, enabled the animal to 
escape into a neutral situation. In experimental 
situations allowing choice between positive (re- 
ward), negative (punishment), and neutral 
situations, neither positive nor negative situations 
alone activated escape into such a neutral situa- 
tion. This escape, apparently, depends on an 
interaction between positive and negative elements. 

As indicated earlier (under "Section of Chemi- 
cal Pharmacology") Dr. Hearst and Dr. Szara (of 
the Section of Chemical Pharmacology) have 
joined in the study of individual differences in 
susceptibility of rats to diethyltryptamine. In- 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



185 



teranimal variation in regard to responses to 
psychoactive agents is a relatively unexplored 
field. It is hoped to pursue these findings further 
using compounds other than the ones so far em- 
ployed. 



Child Research Branch* 



SURVEY OF DEVELOPMENTS 

1959 was a year of crisis and change for the 
Branch. In June 1958, Dr. Eedl had announced 
his forthcoming departure. After a careful re- 
view of the studies nearing completion, and con- 
sideration of the developing needs of the total 
clinical investigations program, it was decided 
to terminate the clinical work of the Branch in 
July 1959. Suitable arrangements were made for 
the patients who had been under treatment, and 
a number of the research staff undertook the re- 
sponsibility of staying on until June 1960, in 
order to complete the working up of some of the 
data previously gathered. 

There were, at the time, two groups of patients 
under study. The one group, which had been 
in treatment for some 5 years, consisted of five 
teen-age boys who lived in the Children's Treat- 
ment Residence and will be hereinafter referred 
to as the cottage or residence group. The other 
patient coterie comprised eight latency-age chil- 
dren who had been admitted in September 1959, 
after a brief previous hospitalization for diag- 
nostic study ; they were housed on "Ward 4E and 
will be referred to as the ward group. After the 
decision to terminate the program was made, all 
these patients were accordingly prepared for dis- 
charge and/or transfer to other institutions dur- 
ing the latter part of the spring. The residence 
patients, now faced with the prospect of a termi- 
nation that they had known must come some day 
but which they had not anticipated quite so 
quickly, showed progressively severe breakdown 
in morale, controls, and overall adjustment. Be- 
havioral upset became rife to the point of becom- 
ing overwhelming; staff morale followed suit; 
and it required a series of intensive staff discus- 
sions followed by confrontation of the patients, 

♦Prepared by Joshua D. Noshpitz, M.D., Acting Chief. 



both individually and as a group, for everyone 
at the cottage to be able to reorient himself and 
face the future in an effective manner. In a sense 
the emotional upheavals in the staff paralleled 
those of the patients on every echelon; it was 
only after prolonged and intensive work on these 
issues that the various team members were able 
to regain their sense of proportion, see the salient 
parts of the reality before them, to master these, 
and communicate them to the patients. 

Administratively, serious problems were faced 
in terms of future placement and followup plan- 
ning for the cottage patients, and a number of 
conferences were held at various levels throughout 
the administrative structure, the upshot of which 
was a decision to provide financial backing for 
placement of three of the youngsters. After care- 
ful consideration of several residential treatment 
centers, it was decided to place three of the patients 
at the Berkshire Farms School in Canaan, New 
York. A contract was let for this purpose by the 
National Institutes of Health and the boys were 
transferred in July 1959. A fourth youngster was 
sent home temporarily pending our finding a place 
for him ; he was finally admitted to the Spaulding 
Youth Center in New Hampshire. The fifth resi- 
dence child was returned to his own home. An 
active followup program is being continued with 
a half-time social worker assigned to the project, 
to make monthly visits both to the parental homes 
and to the current placements of all the boys. 

The ward program terminated somewhat less 
traumatically, since at the time of admission, the 
patients had been informed that they would be 
staying only until the coming summer. As sum- 
mer approached, most of them were able to accept 
the fact of the termination without as severe a 
degree of disturbance. It was, nonetheless, a diffi- 
cult episode for the staff who had been deeply in- 
volved in the project. Many of the staff left at 
the time the patients departed, some were trans- 
ferred to other services, while 11 stayed on to work 
on the data that had been gathered over the pre- 
ceeding years and to prepare it for publication. 

This work has proceeded and continues at pres- 
ent. The team structure is that of four full-time 
and one part-time senior researchers, three full- 
time and one part-time junior researchers, a half- 
time social worker, and two secretaries. Oc- 
casional meetings are held. Dr. Redl returns for 



186 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



regular consultations with each staff member. 
Some consultation takes place with former staff 
members, thus making it possible to amplify the 
recorded material in a useful way. 

RESEARCH DEVELOPMENTS AND 
TENTATIVE FINDINGS 

Projects Completed and Reported 

Individual Therapy and Psychopati-iology. Two 
different styles of therapy were pursued in our two 
separate settings during the early part of the year. 
In the cottage, the five boys who had been in in- 
tensive, f our-times-a-week individual therapy con- 
tinued and terminated this therapy. On the ward, 
the ward group which had been in a situation- 
geared type of therapy showed increasing readi- 
ness to adapt to, to accept, and to respond to this 
approach — moreover, in some cases, they began to 
orient themselves toward the ward therapist in 
such a manner that they were in effect converting 
our original structure into a more formal type of 
individual psychotherapy, very largely by their 
own requests and attitudes. For example, they 
might insist on seeing the ward therapist only in 
his office, they wanted regular appointments, and 
they wanted him to avoid otherwise entering into 
their ward life. It was difficult to avoid the im- 
pression that the youngsters were able to make a 
remarkably rapid induction into a psychothera- 
peutic relationship in spite of very severe char- 
acter problems. It will be important to reexamine 
and retest, in other settings, the possibility of 
utilizing such a method as an induction technique 
for the initiation of psychotherapy with hard-to- 
reach patients of this type. Little can be said 
about the possible long-range benefits of such an 
approach — the period of time the patients were 
in treatment was too brief. Our effort was in the 
nature of a pilot study. 

Returning for the moment to the individual 
therapy with the cottage boys, the aspect of the 
interdigitation of this therapeutic process with the 
on-going ward life demanded the attention of a 
number of the investigators, including the cottage 
mother and two of the therapists. A paper by 
Kitchener, Sweet and Citrin, which was presented 
at the orthopsychiatric meeting in 1959, traced 
out in meticulous detail the simultaneous working 
of parallel themes with the therapist in the play- 



room and with significant ward personnel on the 
ward. Attention was addressed to the phenom- 
enon of breaks which occurred in the behavioral 
and therapeutic relationships and the ensuing 
hostile flood which submerged the entire observing 
ego. The egodynamics were described with em- 
phasis on the precariousness of primary-object 
relationships; the inability to endure tension be- 
cause of the sense of unbearable helplessness and 
the certainty that no future gratification can be 
trusted ; the sense of profound emptiness ; the in- 
satiable oral needs with the accompanying fear of 
their seduction, hunger, and destructiveness ; the 
tendency to project the oral sadism and view the 
world as devouring ; the intense castration anxiety 
along with the associated fear of the loss of inner 
substance. The resultant ego structure is one in 
which the acting out has the defensive function of 
denying the dependency and fear of disappoint- 
ment and where it becomes imperative to maintain 
an illusion of omnipotence. Hence, projection, 
magical thinking, and conversion of passive to 
active are routine. Rapid shifting occurs in the 
level of ego functioning ; the observing ego is not 
given much opportunity to develop or to play a 
role in personality actively. The capacity to learn 
from experience is thus markedly limited and 
thought plays but a small part in the patient's 
adjustment. Appropriate management tactics 
were described starting with the recognition of the 
necessary reliving and recapitulation of the most 
infantile layers of the patient's experience around 
incorporation, object loss and delay of impulse. 
Two technical developments were presented the 
first of which concerns the management of aggres- 
sion. In this area, techniques which permit the 
therapist to join in the support and defense help 
realine the child's collapsing ego and are more 
important than interpretation or prohibition. 
One way to achieve this is to turn the emerging 
aggression into a quasi-game. Another way is to 
feed the child's narcissism at a moment when he 
seems frightened. On the other hand, when the 
aggression is used in the form of manipulation, 
i.e. when the ego is pathologically strong rather 
than pathologically weak, it is important to em- 
ploy interpretation or physical limit setting. The 
second development concerned choice in interpre- 
tation. It is essential that the interpretation be 
geared to the phase of treatment. Thus, when a 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



187 



child is communicating primarily through motor 
behavior, interpretations must be accompanied by 
or made through concrete behavioral responses. 
When a child has improved to the point where he 
can utilize symbolic communication, then symbolic 
gestures of one sort or another should be the rule 
for the therapist too. Only later in treatment 
when verbalization becomes important to the 
child, can interpretive efforts of a verbal kind 
impinge effectively. Readiness to shift among 
these levels as the child shifts during any treat- 
ment hour is of the essence. Many similar devices 
need be used by ward personnel with a parallel 
respect for the shifting states and the precarious 
ego situation of the patients. The confusion that 
may in time take place between significant ward 
persons and therapists is considered an expression 
of the patient's finding uniform respect for and 
response to his treatment needs. 

Problems of Technique. The approach to pa- 
tients through the life-space interview as con- 
trasted to individual therapy received its most 
thorough explication and documentation during 
the final months of the project. Many hours of 
life-space interviewing with the ward patients 
who were not in individual therapy served to 
demonstrate the readiness of some of these young- 
sters to move from concrete discussions of specific 
behavioral events and upsets into more family cen- 
tered and conflict-oriented areas. Such conversa- 
tions would sometimes progress into classical- 
seeming psychotherapeutic interviews. The cry: 
"Take me to the playroom" became a popular and 
frequent one to be directed toward the ward doc- 
tor. A major factor in this seemed to be the omni- 
presence of the ward doctor who was often there 
evenings and weekends so as to be continually 
available to pick up on moments of anxiety, lone- 
liness, or simple need for some gratification as well 
as to handle the more intensive behavioral upsets. 
In addition, a mode of approach was employed in 
which ward personnel often joined the thera- 
pist in these interviews so that what might other- 
wise have been complex and tricky stories to get 
straight, now became sharply focussed, accurate 
accounts of what went on with the full details im- 
mediately available, and with the possibility of the 
interpersonal relationships that had been dis- 
turbed by the incident receiving effective and 
ready clarification. This opened an entirely new 



dimension to the patient's perception of staff 
people individually, and of the interacting adult 
world collectively, and ofttime seemed to have 
dramatic and influential effect. Aside from its 
therapeutic aspects, the training value of this pro- 
cedure is very great; the changes in style and 
technique and the growth in professional maturity 
of the staff members were marked. 

Problems in Ego Structure. Two areas that re- 
ceived major thought during the first half of 
1959 were those of diagnosis and ego identity. 
The first involved the notion of making diagnosis 
a composite of several terms, i.e. each diagnosis 
would be a sentence composed of a number of 
clauses, rather than a single descriptive phrase of 
two or three words. Each part of the sentence was 
scheduled to be connected with a definite set of 
categories and the categories were in turn, the psy- 
chological past, the identity structure, the impulse 
orientation, and finally, the symptomatic behavior 
displayed by the particular patient. Various sub- 
categories under each of these, such as, overstimu- 
lated, phantasy-oriented, erotic-aggressive, and so 
forth were defined, and various formulations were 
then possible to describe particular manifestations 
in specific children. This set of diagnostic cate- 
gories was developed exclusively in connection 
with the hyperaggressive child; it is hoped that 
specialists in other areas of childhood disturbance 
might also develop appropriate category classes, 
so that the possibility would emerge to put a num- 
ber of such category sets side by side, and winnow 
their essential similarities and differences with an 
eye towards developing a more significant set of 
diagnostic terms. 

As a derivative from this essay, the categories 
applied to the psychological past were in turn 
viewed in their relationship to the development of 
antisocial behavior in adolescence. The psycho- 
logical events ensuing at puberty were viewed as 
intruders on a scene that had been prepared for by 
a process of overstimulating, depriving, or over- 
gratifying the child in the preschool period, with 
the inevitable consequences that ensued. It was 
anticipated that this type of approach might serve 
to complement the sociological approach to delin- 
quency where it is possible to account for a delin- 
quency-prone population, but more difficult to ex- 
plain why only a small percentage of such a 



556044—60- 



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188 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



population actually becomes involved with the law. 
If necessary psychological factors which may have 
been present in the past of the particular child 
who becomes delinquent can be seen as additions 
to the sociologic vectors, both functioning as nec- 
essary, but neither as a sufficient cause, a more 
difinitive system emerges. 

Similarly, the question of ego identity — what it 
meant, how it functioned, what its psychological 
and dynamic structure was, its relationship to the 
defensive aspects of personality, some of the forms 
it might take in latency children — was explored 
and will continue to be studied. In particular, the 
role of the choice for or against one's own chrono- 
logical age was viewed as an essential polarity of 
identity-formation in childhood. Other such po- 
larities included the choice of the reality-fantasy 
issue, i.e., was someone a real person or a make- 
believe person ; and the choice between the covert 
and the overt, i.e., was the child "really" to be a 
concealed individual beneath quite a different out- 
ward appearance, or was he to be someone who was 
what he was on the surface. Finally, there seemed 
to be a choice between having any identity at all 
or no identity (which amounts, in effect, to the 
choice between nonpsychosis and psychosis). 
Some children were so vague, so amorphous, so 
lacking in outline, as to be labeled "diffuse" in our 
identity category. 

Milieu Therapy and Life Space Interview. In 
attempting to define what milieu therapy and 
residential treatment needed to differentiate them 
from other types of housing of disturbed children, 
the simultaneous employment of ego support and 
ego analysis as technical modalities in creating a 
therapeutic milieu were described. The notion of 
how the milieu can come to grips with a symptom 
without allowing the child too easy gratification of 
his acting-out on the one hand, or attacking him 
and increasing his defensiveness on the. other, were 
exemplified. The need for the milieu personnel to 
respect the particular ego configuration of the 
youngster and give strength to his defenses when 
they were weak and when anxiety was high, was 
studied at some length and the correlated approach 
of interpreting symptomatic acts as defenses when 
these were strong was equally stressed. These two 
techniques alternating back and forth with the 
same child were seen as essential methods of work- 



ing with him in the environment. Thus, a theory 
of residential treatment built on ego analysis and 
ego support was developed. 

A group of clinical studies on experiences with 
self mutilation in children was presented at a 
symposium on this subject at the orthopsychiatric 
meeting in 1959. Generally, these pointed up the 
interpersonal meanings as well as the masochistic 
and subjective aspects of this behavior. Self- 
mutilating behavior was observed to be : an attack 
on the person of the therapist in the case of a child 
prone to symbiotic fusion experiences ; a symbolic 
act of control at a distance in youngsters who were 
not quite so primitive; a manipualtion of and a 
punishment for therapists ; a coercive for achiev- 
ing or forcing reactions of some kind out of the 
staff; and it might also have the value of a sort 
of masturbatory pleasure. Numerous case illus- 
trations were cited. 

A review of the thinking that had gone into the 
construction of the treatment cottage and of some 
of the problems that had developed around the use 
of space was also the subject of a paper. The 
notion of which elements in the spatial arrange- 
ments around a child's life can be combined and 
which need to be separated was examined at some 
length, and the impact of architecture on the treat- 
ment process was touched upon. In particular, 
the roles of staff residence space and child resi- 
dence space in terms both of the need for nearness, 
the nature of distance, and the kind of distance 
required were observed both for their advantages 
and disadvantages. 

An additional contribution with important tech- 
nical implications was an account of the develop- 
ments around informing the cottage patients that 
the program was about to end. When the young- 
sters learned of their impending separation and 
termination, a highly anxiety-charged situation 
deA r eloped which in turn generated a series of tac- 
tics for countercontrol. This was described in 
some detail: such elements as surprise, concerted 
adult action, group plus individual interviewing, 
intensive and protected programming during the 
crisis time, active limit setting, and, above all, re- 
current interpretation, were blended into a com- 
bined approach with good success. This experi- 
ence has many practical implications for the 
management of institutional crises. 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



189 



Learning Disturbances. The modifications in 
school methods necessary for disturbed children 
were carefully studied. In one paper a five-step 
series of modifications from normal school proce- 
dure was described involving (a) shortening the 
small group school sessions to fit the task involve- 
ment span of the group, (b) adhering to the lowest 
common denominator of academic working level, 
(c) choosing materials which were as free as possi- 
ble from the predominant problems of the chil- 
dren, (d) the design of procedures that were not 
ambiguous or dependent on a child's self control, 
and finally, (e) the attempt to demonstrate an un- 
derstanding of the child's pathology while simul- 
taneously discouraging the manifestations of it in 
the classroom. 

In addition to these technical procedures, their 
counterpart — the impact of work of this sort on 
the teacher's personality — was examined by one of 
our teachers and the nature and kind of growth 
that emerged were explored. In particular, an 
account was given of the major alteration in phi- 
losophy that had taken place between the time that 
the first long-term group had been initiated into 
the classroom, and the time when the work with 
the second long-term group was begun. This 
change involved moving from a position in which 
the teacher attempted to follow the patient at the 
youngster's own level, wherever that might be, to 
a position in which the teacher defined a certain 
range of limits within which the youngster must 
operate with the understanding that if the young- 
ster overthrew the traces or stepped out of these 
limits, he must leave the classroom until he could 
accept this configuration and return. This 
change had profound implications for the entire 
learning process, the milieu, and the adjustment 
of the teacher. To begin with, the child was 
confronted with a much more clear-cut learning 
task with which he could cope either by refusing 
or rejecting or fighting against it some way, or by 
accepting it — in each case he always knew exactly 
what was expected of him, and where he was. 
Secondly, the milieu knew that the school was 
going to be rather taxing and specific in its de- 
mands and that youngsters might come out of 
school tense and pent up from having had to con- 
trol themselves in order to meet the school de- 
mands — this made necessary the planning of much 
activity after school. Third, the teachers came to 



view themselves more specifically as educators 
rather than as teacher-therapists and could 
work within a structure that permitted them 
to apply their teaching skills in a specific and 
articulate way. It will probably remain an area 
of contention in the field for years to come, 
whether in general the more structured school is 
better for a child within a residential setting, or 
whether the residential school should be primarily 
and essentially a flexible instrument which views 
education as part of the treatment process and 
hence bends as the child bends. In any case this 
was the evolution that took place here, and seemed 
to have beneficial results. 

The fact that the "ideal" school conditions under 
which the teachers theoretically worked here did 
not free the school from the basic problems that 
every school must face was reported in one com- 
munication. These basic issues were : the need to 
cope with groups, the need to cope with the body 
of theory and techniques available to teachers, and 
the need of the school to live within a certain com- 
munity and reflect the aims and attitudes of that 
community. Inevitably, intricacies in operation 
will arise on each of these levels, regardless of 
whether one is working in a school with tiny 
classes under the most carefully structured condi- 
tions, or whether one is working with huge classes 
and under the most diffuse conditions. The fact 
that the school is structured in a hospital and must 
thus come to grips with the attitudes, manner of 
life, values, and personnel structure that are nor- 
mal to this type of social community places, on a 
hospital school, very special problems, different in 
degree but not in kind from those of the school in 
any other community. 

The problems encountered in the transfer of 
the cottage patients from the in-hospital school 
to the public schools were studied in detail, and 
some generalizations, useful to any workers in the 
field who face the problems of entering institu- 
tionalized children into public schools were devel- 
oped. In brief, three major issues were defined: 
(1) the decision as to when a child can begin to 
benefit from schooling outside the treatment insti- 
tution, (2) the decisions that must be made around 
the planning for and selecting an appropriate 
school, and (3) the decisions about the quality and 
quantity of power and role distribution between 
school and institution to make it possible for the 



190 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



child to utilize the school experience. In connec- 
tion with the first of these decisions, it was noted 
that the direction in which the child was moving 
was more important than his current situation at 
any given moment, i.e., readiness for new experi- 
ences is often seen in potential ways by the amount 
of development the child has been able to make 
and the tendencies discernible in his overall ad- 
justment rather than by how well he may do on 
any particular day within the institution. The 
obvious clinical issues, of course, must be consid- 
ered, such as how he behaves, what he does in 
therapy, and how he handles community activities. 
In particular, the school record prior to admis- 
sion must be analyzed for behavior crises and 
issues that occurred there. Attitudes toward the 
teacher are subjects for evaluation, as is the child's 
capacity to adjust to a new pupil group. Routines 
need to be weighed and the youngster's ability 
to adapt to them as well as the use he makes of 
ordinary school methods, all must be measured in 
sum. With this in mind, a simultaneous evalua- 
tion of school and child was undertaken and a 
number of criteria were developed. Some of these 
were : the attitude of the staff of the school under 
consideration toward psychiatric treatment within 
that school ; the nature of staff personalities who 
would be involved with the student; the physical 
plant of the school; its geographical location in 
terms of how hard it is to bring the child there or 
for the cottage staff to get there in a hurry if 
necessary ; the amount of crowding and nature of 
the session pattern at school; the relationship of 
that school to the child's family with special 
weight on previous contacts with parents or with 
siblings ; the nature of the social structure of the 
school as compared to the social group of the 
child ; the potentials for social groups within that 
school to become pathological; the age grouping 
of the children who are in the patient's grade ; and 
the complexity of the school structure, e.g. junior 
high vs. grade school, to which the youngster must 
return. The attiude of the principal was of 
prime importance, and some observation of the 
teaching methods and classroom manner of all 
the potential teachers in advance of allowing a 
child to contact them was considered a sine qua 
non for any possible success. 

All the above factors are of the essence in the 
resolution of the third major issue, i.e., the ar- 



rangements between school and treatment institu- 
tion. A very carefully tailored program was ar- 
ranged for each child at school, with the appro- 
priate courses worked out well in advance. One 
parent surrogate from the institution represented 
each child at the school. In addition an educa- 
tionally sophisticated representative of the treat- 
ment institution kept in regular contact with the 
school in order to consult with teachers and prin- 
cipals. Opportunities were afforded for continued 
discussion of all issues ; there were regularly sched- 
uled institution-school conferences designed to dis- 
cuss homework and community expectations. 
Special tutoring was also a feature of the work. 
Important contacts were made with student coun- 
selors in all schools and with the student discipli- 
narians in junior high school. Reports from 
school were obtained for inclusion in the institu- 
tional records, and planning conferences for grade 
placement the following year were initiated early 
in the year in order to facilitate the patient's pro- 
motion with a minimum amount of stress and 
strain. 

Behavoiral Measurement and Assessment of 
Change. A continuing group of studies had been 
devoted to surveying the social interactions of the 
cottage boys and many codeable observations were 
made on how they related themselves to others and 
how others related to them. In an earlier study, 
the interpersonal behavior had been observed at 
two periods a year and a half apart and the 
changes over these periods were examined. Two 
studies on this data were completed in 1959. In 
one the influence of various social settings on the 
interpersonal behaviors was explored, and it was 
established that knowledge of the social setting in- 
creased the variations in the types of behaviors 
observed on the part of the boys. What was not 
anticipated, however, was that the interactive ef- 
fect between the child and the settings revealed 
much more information about the youngsters' be- 
havior that did the sum of all the independent 
components of behavior that were observed sepa- 
rately. It was concluded that the behavior evoked 
by a particular setting was related more to the 
personality of the particular child than to any 
other single factor. As they grew older and con- 
tinued in treatment, the children's ability to make 
differentiations and variations in social behavior 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



191 



increased, this being dependent upon the nature of 
the setting. One of the important implications of 
this for further research is that it seems likely that 
observers would differ somewhat in their estimate 
of improvement depending on their locales of ob- 
servation. In order for such an entity as improve- 
ment to be studied adequately, there would have to 
be a representative sampling of a variety of situ- 
ations in which the behaviors took place. 

The second major study compared the social 
behavior of the patients at the two phases at which 
they were observed with that of two groups of 
well-adjusted children who acted as controls. 
These control normals lived for brief periods in 
the same hospital ward as the patients and were 
observed in identical situations. Thus, with age 
and situation controlled, an attempt was made to 
decide whether the changes that occurred in the 
patients were due to the effectiveness of the treat- 
ment program or rather to maturation. It was 
found that a certain increase in appropriateness 
of social response observed in the disturbed chil- 
dren's peer relations did seem to be related to treat- 
ment rather than to age changes. In terms of 
their behavior toward adults, the children tended 
more and more to approach that of the normal 
controls. These favorable changes were also 
judged attributable to treatment. On the other 
hand, the disturbed children who had shown less 
overt dependent behavior toward adults when they 
were younger, came to exhibit increasing de- 
pendency toward adults as they matured. Since 
normal youngsters tended to become more inde- 
pendent as they matured, this too was viewed as a 
direct effect of treatment. It was concluded that 
the forces for change in social behavior are derived 
from adults rather than from other sources. 
Normal children tended to differentiate among 
social settings more than did the disturbed chil- 
dren, and their behavior was more predictable. 

In general, both the normal and the disturbed 
children showed the same behavioral tendencies 
toward specific settings whether it was mealtime 
or play. The relationships between the behavior 
evoked by a particular setting and the personality 
of a particular child was a constant for the 
normals as it was for the disturbed children. This 
in a sense was the concluding work in this whole 
skein of observations on the interactions of chil- 
dren and adults within the treatment settings. 



The use of the normal controls enabled the investi- 
gators to establish more definitively both methods 
of observations as well as an evolution of the 
meanings of improvement for these youngsters. 

A derivative study that emerged from the ob- 
servations made on the control normals had im- 
plications for a psychodynamic behavior theory. 
Normal ego growth was seen as something more 
than an absence of pathology but rather as a series 
of positive achievements meriting descriptive 
formulation, and, in time, genetic investigation. 
Such elements as the development of time prospec- 
tive, the sense of trust in the future, the sense of 
objectivity (i.e. detachment from narcissistic equa- 
tion of self and object) the ability to delay, the 
tolerance for frustration, the realistic perception 
of cause and effect, the capacity for initiative and 
taking responsibility, the search for mastery, and 
the sense of self-esteem were categorized as areas 
for observation and measurement. A number of 
achievements of the normal latency child were ob- 
served and comments were made on the reflections 
of ego growth in the use of speech, in the develop- 
ment of peer relations and ego controls, and in the 
techniques employed by such boys to control their 
own impulses and to advance support to each other 
in the face of tempting and exciting situations. 

Studies of improvement were also undertaken. 
One investigator recorded the comments and ob- 
servations of various staff members about the state 
of each of the residence patients at 6 months prior 
to the move from ward to cottage, and then again 
6 months after the move. In tabulating this in- 
formation, five areas of improvement emerged. 
The first lay in the extension of ego horizons of 
the patients, so that totally new areas of function- 
ing came into view. The second was the use of 
withdrawal as a healthy coping device in contrast 
to the acting-out-impulses behavior of the past — 
it was an interesting phenomenon that what in 
normals might have been considered pathological 
behavior, with these patients was used in the serv- 
ice of increasing mental health. Third, there were 
shifts in balance between individual- and group- 
centeredness ; instead of the youngsters banding 
together and operating like a gang who are being 
oppressed and must resist the adults, each one be- 
gan to individualize, differentiate and seek his own 
goals, and to express his own needs quite apart 
from those of the others. Fourth, there were 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



changes in sensitivity to danger potentials; the 
youngsters became aware of problems earlier, and 
recognized and responded to each other's recog- 
nition of them more quickly and subtly. And, 
finally, there was a development of a concept of 
self in such areas as sense of responsibility, pride, 
identity, humor, objectivity and sense of time 
which marked the progress of each of the 
youngsters. 

A second study on improvement sought to evalu- 
ate the meaning of the term, to measure the im- 
pact on the staff of the phenomena of improve- 
ment, and to record some of the resistances and 
anxieties as well as the downright opposition that 
developed to the changes taking place in the 
patients. This latter was called the phenomenon 
of "improvement panic." It occurred when a staff 
which had been geared to coping with and re- 
sponding to the most seriously disturbed behavior 
began to find that they were in fact encountering 
less disorganized and explosive situations than 
previously. As a result, such normal staff activi- 
ties as recording and discussion showed up mark- 
edly; there seemed to be nothing to write and 
nothing to say about the less pathological patients. 
There was an increase in covert interdisciplinary 
tension; much argument about whether the 
patients were really getting better or whether this 
was just a naive, over-optimistic statement; and 
violent rejection of any attempt to observe the 
possible improvement at all closely. This was 
carried to the point of outright anger at the entire 
project together with a nostalgia for the "good old 
days," when things had been "different" and 
very difficult. The narcissistic investment in the 
child's pathology, the feeling of being needed by 
him, the awareness of one's own errors that a 
study of his improvement might reveal, were 
among the factors that appeared to be operative. 
Even the term improvement itself had many 
meanings. It might mean a change in overall 
mental health; it might mean alteration in the 
individual functioning of the person as a human 
being; it might mean an alteration in the ease 
with which one could live with a patient ; it might 
simply mean an abstract function with no par- 
ticular connection with behavior. In any case, the 
impact on the staff was painful, and sometimes 
more painful on the more sophisticated than on 
the less thoroughly trained members of the team. 



Of particular concern was the question of whether 
or not a given "bit" of improvement was "real" 
or whether it was merely a defense against treat- 
ment. Moreover, even if present in any one field, 
was it ready for transfer to other areas ? Again, 
the question was raised as to whether improve- 
ment meant that the child would not break down 
again or whether the bases on which the progress 
rested were "genuine." 

Some of the clinical changes that occur in 
patients when they start to improve were reviewed 
as well as some of the additional problems they 
might have to face ; e.g., in coping with newer and 
more difficult situations, there would be greater 
anxiety about failure and about loss of control. 
The many problems the youngsters encounter with 
their peers, once they begin to improve, and their 
sensitivity to adult reactions, with the consequent 
use of their newfound abilities as bargaining and 
manipulative tactics also alter the acceptability 
of and the staff response to their improvement. 
To improve means to have more choices than be- 
fore, which is in itself a source of turmoil — 
particularly if the adults now begin to leave the 
youngsters on their own more, and thus implicitly 
demand more of them. Inevitably the patients 
make some wrong decisions and must face the con- 
sequences of these. The clinical staff must cope 
constantly with the temptation to overexpect and 
overexploit the improvement and to make quick 
deals, so to speak, with a child whose improve- 
ment is really more of a mask and a defense 
than it is a genuine growth. The youngsters 
should not be made to feel that the response to 
their improvement is a reward for having been 
"good" or a privilege they have been granted. 
Without being indifferent to their growth, one 
tries to communicate to them that the improve- 
ment is something that is accepted just as grow- 
ing taller would be. It is readiness rather than 
privilege that is the criterion for providing new 
opportunities. 

On their side, staff members have difficulty re- 
pressing their own desire to gloat over success. 
Rivalry may develop among the various "fields" 
or disciplines and tends to be augmented by these 
changes, and by determining who is responsible 
for the improvement and where is it seen best. 
There are some real dangers in this since the treat- 
ment atmosphere can be turned into a reward and 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



193 



punishment type structure, and a system of caste 
may develop with the inevitable outcasts. This 
may emerge all too readily around the fact that 
some children are improving and other children 
are not. At the same time, the need for a more 
complex program emerges, so that the varying 
achievement levels of all the children can be met 
instead of some being gratified and some deprived. 
In the face of these difficulties, it is often dif- 
ficult to decide whether a particular upset means 
that a child is regressing, or whether he is merely 
experiencing the pangs of trying to cope with a 
necessary new experience. One must, therefore, 
take calculated risks and be ready to accept the 
public reaction to the inevitable failures that will 
ensue. This study casts considerable doubt on the 
value of any type of intensity scale of skills, traits 
or personality characteristics as a measure of im- 
provement. A serious weakness in our current 
descriptions is that the language depicting the 
psychological ingredients of settings and situa- 
tions is very weak compared to the language we 
have to describe the changes in behavior itself. 

The Biographical Studies 

The major area of dedication of the final year's 
work of the Child Eesearch Branch lies in a 
series of six biographies, one for each of the resi- 
dent patients, which are currently engaging the 
energy and the attention of the senior research 
staff. It is expected that these biographies will 
eventually be brought together into a single vol- 
ume which will be a formal statement of the lives 
and of some of the clinical thinking that went 
into the treatment of each of these boys. A 
second major study now under way is the defini- 
tive writing-up of the material on life-space inter- 
view. Here we will attempt to summarize all 
the various experiences we have had with the 
modality of treatment, to categorize types of life- 
space interview, to relate this interview to the 
therapeutic interview, and to explore the differ- 
ences between the earlier style of life-space inter- 
view initiated with the long-term group and the 
later style developed on the ward with the eight 
younger patients. 

One investigator is making an intensive exami- 
nation of the learning problem of one of the long- 
term patients, will attempt to relate this to the 
youngster's psychopathology. He hopes to cor- 



relate the learning difficulties as they were encoun- 
tered day-by-day in the classroom with the psycho- 
therapy hours and the content and the style of 
adjustment in the therapy. It is difficult at this 
point to predict exactly what will emerge from so 
careful a study with the large amount of data 
available — certainly the oft-discussed question of 
the interaction between psychopathology and 
learning difficulty should receive a searching and 
throughly documented explication. 

A handbook on residential treatment based on 
cumulative experiences here is being outlined. It 
will review some of the broader outlines of treat- 
ment methods within a residence, the philosophy 
of treatment, the techniques, the problems, di- 
agnostic issues, staff training, and some of the diffi- 
culties and attempted solutions that have emerged 
in the course of the interactions. 

A number of less ambitious projects are also in 
prospect. Several of the boys have sustained 
losses of important relatives during their time 
in residence, and the record of their reactions is 
being assembled in order to study something about 
the meaning of such a loss to this type of child 
with particular emphasis on the forms of expres- 
sion of this reaction throughout the totality of 
the child's life. 

In reviewing the way some of our patients came 
to us, and some of the things that happened to 
their families, both before and during the course 
of treatment, one of our staff was impressed by the 
way that the values of the mental-health personnel 
who handled these families and patients at one 
point or another became entangled with the issue 
of the management and treatment of the patients. 
It is anticipated that a report of these observations 
will be presented, and that they will prove useful 
to social agencies in dealing with disturbed 
families. 

Another book in prospect is the history of the 
educational aspect of the project, i.e., the record of 
the total experience in attempting to establish the 
school, the changes in method, the philosophy, and 
a thorough reworking of the meaning of the edu- 
cational issues within the total undertakinp-. 

Yet another report is being devoted to a history 
of the project as a whole with a careful examina- 
tion of its origin, many of the events that have 
played a role in it, the administrative issues in- 
volved in its structure, the many clinical events 



194 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



that determined its form, with particular emphasis 
on the theoretical implications of the development 
of this type of social structure within a larger 
institution. 



Laboratory of Clinical Science 

The laboratory of Clinical Science was estab- 
lished to straddle what might have become a gap 
between the basic disciplines, especially the bio- 
logical fields of biochemistry, physiology, and 
pharmacology, and the problems of psychiatric 
disease. Its division into basic and clinical sec- 
tions represented the stake which each of these 
programs has in the functions of the Laboratory, 
but, although the scientific work has represented 
a broad spectrum of activity from studies on 
patients with psychiatric disorder, on normal 
volunteers or on nonclinical and rather basic prob- 
lems, there is no precise relationship of the sci- 
entific activities to the administrative division. 
In fact, the extent to which the clinical-basic divi- 
sion is not clear represents the success which the 
Laboratory has had in breaking down the concep- 
tual barriers which often separate these two ap- 
proaches. For purposes of this summary, the 
work of the Laboratory in the past year may be 
divided into certain problem areas : schizophrenia, 
aging, experimental allergic encephalomyelitis, 
sleep, and specific problems of metabolism related 
to the nervous system or behavior. 

SCHIZOPHRENIA 

The Laboratory has continued its major pro- 
gram of investigation into the possible role of 
biological factors in the etiology and pathogenesis 
of schizophrenia and their interaction with social 
and psychological factors. The Section on Psy- 
chiatry, in selecting patients for Laboratory 
studies, had previously made a broad survey of 
the family histories of male patients diagnosed as 
schizophrenics in Maryland and District of Co- 
lumbia public hospitals. This survey provided an 
opportunity for an analysis by Dr. William Pollin 
and his associates of the distribution of schizo- 
phrenia and other forms of mental illness in the 
families of schizophrenics. This revealed a pre- 
ponderance of schizophrenia in the mothers, as 

♦Prepared by Seymour S. Kety, M.D., Chief. 



opposed to the fathers, of such patients and was 
further supported by an extensive analysis of data 
in the literature. Although this appeared to sup- 
port the sociologically based theories of the role of 
maternal influence in the development of schizo- 
phrenia, further analyses of alternative explana- 
tions revealed that the findings were compatible 
with a considerably greater marriage and fertility 
rate found among schizophrenic females in con- 
trast to males. 

The Section on Psychiatry is presently engaged 
in a careful study of the life situations of the se- 
lected group of patients under investigation by 
the Laboratory in comparison with their non- 
schizophrenic siblings, in an effort to elucidate the 
special and perhaps highly individualized psycho- 
social factors operating before the development of 
the mental disorder. 

Among the attractive hypotheses recently for- 
mulated to account for schizophrenia in biochemi- 
cal terms, perhaps the most interesting is that 
based upon a postulated disorder in the metabolism 
of circulating epinephrine, entailing production of 
possibly psychotomimetic substances such as 
adrenochrome. By virtue of the recent excellent 
work of Dr. Julius Axelrod in elucidating this 
hormone's normal metabolism, the Laboratory was 
uniquely equipped to test that hypothesis. Drs. 
Stephan Szara, Julius Axelrod, and Seymour 
Perlin, more than a year ago, had ruled out the 
presence of abnormal or even detectable concentra- 
tions of adrenochrome in the blood of schizo- 
phrenic patients, and, at the same time, Dr. Roger 
McDonald and his associates had demonstrated 
that the reported rapid in vitro oxidation of epine- 
phrine by the serum of such patients was the re- 
sult of a dietary deficiency of ascorbic acid. An 
extensive study of the metabolism and the physio- 
logical and psychological epinephrine infused into 
a series of normal controls and schizophrenic pa- 
tients was undertaken collaboratively by a number 
of investigators in the Laboratory. Dr. Jay Mann 
has found that the overall rate of metabolism of 
this hormone as judged by blood levels achieved 
and the rate of their decay was identical in the two 
groups, while Dr. Elwood LaBrosse has demon- 
strated that the pathways of metabolism and the 
metabolic products of epinephrine were the same 
in schizophrenics as in normal man. Studies by 
Dr. Philippe Cardon on the cardiovascular effects 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



195 



of infused epinephrine, by Dr. Louis Sokoloff on 
the blood-glucose response, and by Dr. William 
Pollin on the mental effects, tended to confirm the 
slight differences previously reported. Although 
the latter findings indicate some differences in re- 
sponse, which are being further studied, the meta- 
bolic studies leave little room for the possibility of 
the generalized disturbance in epinephrine metab- 
olism, which had been postulated in this disorder. 

Because of suggested possibilities of a disturb- 
ance in histidine metabolism in schizophrenia, Dr. 
Donald Brown, of the Section on Biochemistry, 
undertook a study of the urinary metabolites of 
the amino acid uniformly labeled with C 14 . Al- 
though this study resulted in a number of signifi- 
cant basic findings which are discussed later, it 
revealed no differences between the normal and 
schizophrenic patterns. 

There is no a priori reason to suspect that if a 
biochemical abnormality exists in some types of 
schizophrenia, it must be a generalized one. On 
the other hand, a number of cogent arguments 
point to the highly differentiated metabolism of 
the brain as more likely to harbor important 
chemical mediators of behavior, both normal and 
abnormal. The inaccessibility of the brain for 
investigation during life makes it necessary to 
devise indirect methods of approach to possible 
cerebral metabolic disorders in schizophrenic pa- 
tient's. Dr. Irwin Kopin has been developing 
highly original, double-labeling techniques which 
may be suitable for the study of the metabolic 
turnover of such substances as serotonin and 
norepinephrine and other amines in the human 
brain. Another approach is to produce certain 
mild alterations in brain chemistry on the basis 
of biochemical theory or of changes shown to 
occur by direct analysis in animals, and carefully 
to study the effect of these discrete chemical 
changes on mental function as determined by 
carefully controlled but intensive psychological 
and psychiatric evaluations. One such study, 
initiated hi the past 3 months by a number of 
investigators of this and other laboratories, em- 
ploys the dietary or parenteral administration of 
certain precursors of possibly psychoactive sub- 
stances which may occur in the brain in conjunc- 
tion with certain enzyme inhibitors which may 
retard their destruction. There has been an op- 
portunity partially to test tryptophan, phenylal- 



anine, histidine, glutamine, glycine, and methio- 
nine with and without the possible potentiating 
effect of small doses of iproniazid in this situation. 
Certain behavioral changes have been observed 
and reproduced, and these findings are behig 
actively pursued. 

The discovery and characterization of the 
significant metabolites of epinephrine and nore- 
pinephrine make possible for the first time re- 
liable measurement of the endogenous production 
of these important hormones, not only in schizo- 
phrenia but in a large variety of psychiatric 
states and situations. Dr. Roger McDonald and 
Dr. Elwood LaBrosse and their associates have 
undertaken the development of simple and reliable 
methods for the quantification of 3-methoxy-4- 
hydroxy-mandelic acid and of metanephrine and 
normetanephrine, respectively, in the urine. A 
method for the first of these compounds has 
already been developed and is being applied in 
studies of schizophrenia and other mental states 
and the effects of certain psychoactive drugs. 

New information on the properties of certain 
drugs extensively used in schizophrenia has been 
obtained in the past year. Dr. Julius Axelrod, of 
the Section on Pharmacology, in showing that 
both chlorpromazine and reserpine speed the de- 
struction of epinephrine in vivo, has demonstrated 
one of the rare biochemical effects which these two 
drugs — both with similar psychiatric effects — 
have thus far revealed to have in common. Con- 
firming this in man, Dr. Roger McDonald has 
shown a sharp increase in the excretion of a major 
metabolite of the catecholamines following a single 
dose of reserpine. Dr. Conan Kornetsky further 
replicated his finding of a differential effect of 
chlorpromazine on the standing blood pressure of 
normals and schizophrenic patients, and plans, in 
his new position at Boston University, to investi- 
gate the possible mechanisms of this action. 

The absence of specific biological criteria in 
schizophrenia is matched by the paucity of reliable 
objective psychometric indices of the disorder. 
Dr. Irwin Feinberg, of the Section on Physiology, 
has carried out a series of studies aimed at differ- 
entiating the mental impairment associated with 
schizophrenia from that associated with non- 
schizophrenic illnesses. He has succeeded in de- 
signing a psychological test on which the per- 
formance of acute schizophrenic subjects differs 



196 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



significantly from that of patients with an organic 
mental syndrome. The test in question, a modifi- 
cation of Raven's Matrices, shows that the acute 
schizophrenic patients make many more unreason- 
able errors than do chronic schizophrenic patients 
or patients with organic mental syndrome. These 
results point the way to a more precise and objec- 
tive characterization of the nature of cognitive im- 
pairment in schizophrenia. The Section on Psy- 
chiatry is continuing its work on the development 
and testing of new methodologies which may pro- 
vide data both clinically and psychodynamically 
meaningful and, at the same time, verifiable and 
quantifiable. 

The manifestations of psychiatric illness, more 
than any other group of diseases, probably repre- 
sent the interaction of a multitude of factors from 
the sociological as well as the biological spheres, 
and a single and sufficient cause for a process like 
schizophrenia is probably not to be expected. One 
of the real values of an intensive interdisciplinary 
study of a selected small sample of patients is the 
opportunity for relating, in the same patient, find- 
ings of one discipline to those of many others. To 
take full advantage of this opportunity, the Lab- 
oratory, with invaluable collaboration by Dr. 
Samuel Greenhouse of the Biometrics Branch, 
NIMH, and by the computer facility of NTH, has 
undertaken a program of data reduction and com- 
parison which will make for maximum utilization 
of the data obtained by the individual investiga- 
tors and their intercorrelation with other infor- 
mation. 

AGING 

Several cogent developments resulted from 
studies by Dr. Louis Sokoloff and the Section on 
Cerebral Metabolism in over 50 normal elderly 
men, carefully selected for their relative freedom 
from the common degenerative diseases of old age 
and functioning competently in their communities, 
which were completed and analyzed in the past 
year, as part of a large collaborative study in this 
Institute. This series showed no reduction in 
cerebral circulation or cerebral oxygen consump- 
tion in comparison with healthy young men, indi- 
cating that the reduction in these functions usually 
found in less carefully selected patients is not a 
necessary concomitant of the aging process. In 
the presence of arteriosclerosis of varying degrees 



there is a decreased cerebral blood flow, a decrease 
in cerebral venous oxygen tension indicative of 
cerebral anoxia, and a somewhat smaller fall in 
oxygen utilization, all of which appear to be cor- 
related with the degree and duration of the arterio- 
sclerosis and the psychological deficit, suggesting 
that one of the primary changes in the mental 
disorders associated in some individuals with aging 
is cerebral circulatory insufficiency and the result- 
ant partial cerebral anoxia. Patients suffering 
from what is known as chronic brain syndrome 
showed a more marked decrease in cerebral oxygen 
consumption compatible with the thesis that this 
syndrome represents parenchymal damage in the 
last stages of progressive cerebral ischemia. 

Drs. Robert Butler and Seymour Perlin of the 
Section on Psychiatry have studied these patients 
from the psychiatric point of view. In addition 
to contributing the psychiatric component of the 
correlations mentioned above, their studies on the 
psychiatric aspects of the aging process have re- 
vealed the importance of the personal meaning of 
psychosocial changes in terms of the individual 
personality as compared with the nature or inci- 
dence of the stresses themselves. The psychologi- 
cal defense mechanisms utilized by the volunteers 
and patients were studied and described in terms 
of their adaptive or maladaptive consequences. 

EXPERIMENTAL ALLERGIC 
ENCEPHALOMYELITIS 

This experimental disorder, produced in guinea 
pigs by the subcutaneous injection of brain tissue 
with certain adjuvants, offers a useful model for 
the investigation of multiple sclerosis and other 
demyelinative or degenerative diseases. During 
the past year, the Section on Biochemistry, under 
the leadership of Dr. Marian Kies, has continued 
its studies on the etiology and pathogenesis of 
this disease. Purification of a water soluble anti- 
gen continues to be of major importance in this 
project. Encouraging results have been obtained 
with chromatography on modified starch columns 
as a means of separating traces of inactive protein 
from the antigenic material. 

Of considerable significance to both patho- 
genesis and treatment of the experimental disease 
are the immunologic results obtained with this 
purified antigenic fraction. All immunologic 
tests on the antigen (skin and corneal hypersen- 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



197 



sitivity, serum antibody reactions) have been 
negative. However, carefully controlled skin 
testing has led to the observation that the disease 
can be suppressed by intracutaneous injections of 
aqueous solutions of the active fraction after the 
initial injection. A study of the significant 
variables in suppression of the disease in this 
manner offers exciting possibilities with regard to 
therapy and prevention of related neurologic 
diseases in humans. 

SLEEP 

This universal and mysterious state is an im- 
portant segment of normal mental function and 
has associated with it the phenomenon of dream- 
ing with certain interesting parallels to schizo- 
phrenic thought processes. Ten years ago, Dr. 
Sokoloff and Dr. Kety and their associates had 
demonstrated that normal sleep was not associated 
with cerebral ischemia, anoxia, nor with the re- 
duction in oxygen and energy utilization associ- 
ated with coma and anesthesia, providing evi- 
dence that sleep consisted of a change in the pat- 
terns of activity in the brain rather than a change 
in their overall intensity. 

During the past year, the electrophysiological 
work of Dr. Edward Evarts and the Section on 
Physiology has been devoted to studies of the 
effects of sleep on the electrical activity of the 
brain. These studies have indicated that sleep 
has different effects on activity in the brain stem 
reticular formation as compared to the cerebral 
cortex. These findings support and extend the 
theories of previous workers (Magoun and 
others) concerning the role of the reticular for- 
mation in the waking state. 

During sleep, potentials evoked by clicks in the 
reticular formation are reduced, whereas cochlear 
nucleus and primary cortical potentials remain 
relatively unchanged. Recordings of single-unit 
activity from the visual cortex show a considerable 
increase in total neuronal discharge during sleep 
as compared to the waking state. These micro- 
electrode studies indicate that during waking there 
is a selective reduction of spontaneous neuronal 
discharge as compared to discharge evoked by 
primary afferent input (electrical stimulation of 
lateral geniculate radiations). This selective re- 
duction of spontaneous neuronal discharge might 



be viewed as leading to an increase in the signal-to- 
noise ratio during waking. This notion involves 
the supposition that the spontaneous discharge is 
"noise" and the discharge evoked by afferent stim- 
ulation is "signal." Such a change in the pattern 
of neuronal discharge may be of importance in 
attention mechanisms associated with the waking 
state. Studies of evoked potentials recorded from 
scalp electrodes in man are to be carried out in 
order to determine the degree to which similar 
alterations of electrical activity may be found as- 
sociated with sleep in man. These studies were 
made possible by an ingenious technique, de- 
veloped by Mr. Robert Cox, for enhancing the 
signal-to-noise ratio in such recordings. 

Dr. Philippe Cardon has continued his interest 
in the physiological and psychological effects of 
sleep deprivation. Subjects whose continuous per- 
formance is impaired show characteristic changes 
in heart rate, respiratory rate and depth, fingertip 
volume and pulse volume, and forearm volume- 
pulse form. These changes occur in the course of 
the test when the subject is not responding to the 
visual or auditory cues presented, and disappear 
when the subject is responding. Thus, there 
seems to be abundant confirmation, at the physio- 
logical level, of the current hypothesis that much 
of the impairment of psychic functioning which 
accompanies sleep loss is due to "lapses" or 
"microsleeps." 

METABOLISM 

Mechanism of Action of Thyroxine. A unique 
feature of the cerebral metabolism is its apparent 
lack of response to high circulating levels of thy- 
roid hormone. An understanding of the basis of 
this unique behavior may reveal information con- 
cerning the metabolism of the brain in health and 
disease. The mechanism of action of thyroxine 
has been under investigation for many decades, 
but thus far a satisfactory explanation of how it 
increases metabolic rate stimulates metamorphosis 
and growth, or causes the many disturbances in 
body physiology and biochemistry in thyroid dis- 
eases, has eluded investigators. 

Dr. Louis Sokoloff and the Section on Cerebral 
Metabolism have continued to make progress in 
their investigations of the mechanism of thyroxine 
action. Their finding last year that L-thyroxine 



198 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



enhances the in vitro incorporation of amino acids 
into protein has been shown to be a definite stimu- 
lation and not a preservative effect. They have 
uncovered evidence of a latent period of action of 
thyroxine in vitro during which a still unidentified 
intermediate is probably formed which is then 
responsible for the stimulation. They have dem- 
onstrated that the formation of this intermediate 
is dependent on the presence of an active oxidative 
phosphorylating system. Their studies with the 
physiologically less-active isomer, D-thyroxine, 
and the physiologically active analogue, L-triiodo- 
thyronine, indicate that the thyroxine effect on 
amino-acid incorporation behaves in a manner to 
be expected of a physiological effect of thyroxine. 
They also suggest that D-thyroxine is physiolog- 
ically inactive, not because the intracellular en- 
zymes involved in the action of L-thyroxine are 
stereo-specific, but because it does not reach the 
enzyme sites when administered into the intact 
animal. Dr. Seymour Kaufman, of the Labora- 
tory of Cellular Pharmacology, has been an active 
collaborator in many of these studies. The find- 
ings of this project represent encouraging progress 
toward the ultimate solution of the mechanism of 
action of the thyroid hormone. 

Metabolism or Epinephrine and Norepineph- 
rine. During the past year, the Section on 
Pharmacology was mainly concerned with studies 
on the metabolism, and physiological disposition of 
H 3 -epinephrine. In collaboration with Dr. Hans 
Weil-Malherbe, the distribution and rate of O- 
methylation of epinephrine were investigated. 
The amine was found to be unevenly distributed 
in various organs and tissues and did not pass the 
blood-brain barrier except to a small extent in 
the hypothalamus. Within two minutes, most of 
the administered catecholamine was O-methylated, 
while part of the hormone was bound by tissue 
constituents and retained in the body for long 
periods of time. 

Dr. Axelrod, in collaboration with Drs. Kopin 
and Mann, reported a new metabolite of epineph- 
rine and norepinephrine, 3-methoxy-4-hydroxy- 
phenylglycol. This compound was shown to arise 
from the deamination of (nor)metanephrine, fol- 
lowed by reduction. Subjects with pheochromo- 
cytomas excreted large amounts of the glycol. 

Inhibitors for catechol- O-methyl transferase in 



vitro and in vivo have been found (pyrogallol and 
quercetin) . Since other investigators have shown 
that these compounds prolong the action of 
epinephrine and sensitize the sympathetic nervous 
system, it would appear that catechol-O-methyl 
transferase is the enzyme chiefly concerned with 
terminating the action of the catecholamine 
hormones. 

Using C 14 - and IP-labeling of various precursors 
and intermediates in the metabolism of catechola- 
mines, a technique is being developed by Dr. 
Kopin which enables, in a single experiment, esti- 
mation of the relative importance of alternate 
pathways of metabolism of one substance to an 
excreted metabolite. The metabolic rate of the 
precursor substance can also be estimated by study 
of the rate of change of the H 3 /C 14 ratio in the ex- 
creted compounds. The effect of various drugs on 
the routes of metabolism of the catecholamines and 
on their rate of metabolism is being studied. By 
this technique, an estimate of the importance of 
the pathways of epinephrine metabolism in man 
has been made. About % of an injected dose of 
epinephrine undergoes methylation while the rest 
is either excreted as such or acted upon by mono- 
amine oxidase. About half of the 3-methoxy-4- 
hydroxyphenylglycol and 3-methoxy-4-hydroxy- 
mandelic acid formed from injected epinephrine is 
formed by methylation followed by deamination. 
In the rat, methylation is of lesser importance, but 
is still a major pathway of metabolism. 

Metabolism or Histidine. Incidental to his 
studies on the metabolism of this amino acid in 
schizophrenic patients, Dr. Donald Brown, of the 
Section on Biochemistry, made a number of con- 
tributions relating to the normal metabolism of 
histidine which dwarfed the initiating study in 
their significance. In collaboration with Dr. Kies,. 
an unstable metabolic intermediate (imidazolone 
propionic acid), whose existence was postulated 
but which had not hitherto been isolated, has been 
stabilized and characterized, and a new metabolite 
present in the urine of man and other species has 
been identified (hydantoin propionic acid). In 
collaboration with Dr. Axelrod, a new methylating 
enzyme has been demonstrated and party char- 
acterized as N-methyl transferase, which catalyzes 
the transfer of CH 3 - from S-adenosylmethionine 
to the imidazole ring of histamine. The presence 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



199 



of this enzyme in highest concentrations in the 
brain suggests the possibility of a significant func- 
tion for histamine or some related amine in central 
nervous function. 

Metabolism of Other Amino Acids. Because of 
the relationship to certain amines which may play 
a central role in the mediation of particular types 
of behavior or emotion, a number of amino acids 
are of special interest to psychiatry. Studies by 
Drs. Elwood LaBrosse, Irwin Kopin, and Shoichi 
Hotta are under way on certain aspects of the 
metabolism of tryptophan, phenylalanine, gluta- 
mine, methionine, and tyrosine, in an effort to re- 
late the differential aspects of their metabolism to 
mental and behavioral state, to dietary intake, or 
to the action of certain psychopharmacologic 
agents which may operate by an effect on such 
pathways. 

ENZYMATIC ACTIVITIES IN BLOOD 

Dr. Roger McDonald in collaboration with Dr. 
Gary Felsenfeld, a former member of the staff of 
the Laboratory of Neurochemistry, has been con- 
cerned with studies on the chemistry of cerulo- 
plasmin. This approach comes as a logical de- 
velopment of previous studies in his Section on 
the possible role of ceruloplasmin in mental dis- 
ease. In the current studies of ceruloplasmin it 
has been shown that copper exists in both the oxi- 
dized and reduced state. Furthermore, when 
ceruloplasmin is actively functioning as an oxi- 
dase, there is an increase in the amount of reduced 
copper present in the molecule. Other aspects of 
the chemistry of ceruloplasmin are presently 
under investigation. 

A second area of study has been by Dr. Franklin 
Evans, of the Section on Medicine, concerning 
serum cholinesterase. In this study, the effects of 
psychotomimetic and psychotropic drugs on two 
forms of human serum cholinesterase have been in- 
vestigated. The difference in the responsiveness 
of the two forms of enzyme to inhibition by the 
psychotomimetic drug, lysergic acid diethylamide, 
was found to deviate from the pattern usually seen, 
suggesting a different mode of reaction. In addi- 
tion, an apparently new and deviant form of 
cholinesterase was discovered in a screening of 
mental hospital patients, although there is no rea- 
son to believe that its presence is related to mental 
disease. 



Laboratory of Psychology* 

As Chief of a combined laboratory, I am again 
submitting a single report to the two Directors. 
It becomes increasingly clear, as the program of 
the Laboratory develops, that the most effective 
long-time research in our area calls for movement 
back and forth from studies of the human to the 
subhuman, from the laboratory to the field, and 
from the clinic to the laboratory. Some of this 
trend is reflected in a constantly increasing over- 
lap between the activities of sections from the two 
programs and within programs. 

Since we have recently rethought our programs 
in the context of long-term plans, it appears wise 
to introduce the description of the progress of 
the various sections during the year against a 
background of what the future program of the 
Psychology Laboratory at NIMH looks like as at 
present projected. 

Although the areas to be listed seem to be quite 
disparate, it is surprising how many interchanges 
and points of mutual fertilization turn up. In 
this respect, more of the outlined program emerges 
as actuality than is apparent on the surface. 

Child Development. This area is likely to see 
tremendous activity and advances during the next 
decade. Child psychology is going through a 
renaissance. I expect that the work which will 
be done by our group will be extended and carried 
out in the context of increasing knowledge of both 
the physical and social aspects of development. 
It is here that the combination of field and labora- 
tory studies both in animal and man are so im- 
portant and for which some of the facilities will 
have to be extended. 

The Psychophysical Parameter. I include 
under this heading a whole area of research which 
our present Animal Behavior and Learning and 
Perception Sections will be involved in during the 
next decade. Psychophysiology and physiopsy- 
chology, psychochemistry and chemopsychology 
(under which might be included pharmacopsy- 
chology) will be going through an unusual de- 
velopment during this time. It is important for 
psychologists working in this area to keep their 
eyes mainly on the psychological aspects. In the 
context of sophistication about physiology and 



♦Prepared by David Shakow, Ph. D., Chief. 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



chemistry they must persist in the careful and 
detailed study of the complexities of behavior — ■ 
learning, especially emotional and social learning, 
perception, emotion and attention, and perhaps in 
time, thinking. Whether the approaches are 
physicopsychological — where lesions, drugs, etc., 
are the independent variables — or psychophysi- 
cal — where psychological conditions are the inde- 
pendent variables — the psychologist's primary job 
is to study the behavior for its tie-in with the 
physical. 

Creativity. This is a new area of endeavor for 
us which I hope will become a major activity 
during the decade because it has such important 
theoretical and practical implications. Our pres- 
ent early thinking is to make a multiple attack on 
the problem of talent and the optimal use of ca- 
pacity; to carry out studies on the motivational, 
environmental and capacity aspects in the clinic, 
the field, and through experimental techniques. 

Psychotherapy. The area of psychotherapy 
viewed as an important highroad to the under- 
standing of personality will continue to be the 
preoccupation of the groups now studying this 
area. Because of the complexity of the data it is 
to be hoped that new techniques of analysis can be 
developed capable of dealing at least in part with 
such multiplex data. 

Schizophrenia. A continued and more extensive 
attack on the psychology of schizophrenia must be 
made. This area has great importance for per- 
sonality theory because in so many ways it pro- 
vides opportunity for study of aberrations from 
the normal which may eventually enable us to 
understand normal phenomena better. In addi- 
tion, of course, this particular disordered group, 
because of its cost, is one of mental health's great- 
est problems. A start has already been made, and 
a broader program is being developed with the use 
not only of patients at the Clinical Center, but 
those at St. Elizabeths and elsewhere. 

Aging. An active program in this area has been 
going on for a number of years. Most recently 
this section's interest in the psychology of aging 
has begun to emphasize higher thought processes 
and continuing effective function in older persons. 
(This latter would tie in with the creativity pro- 



gram.) This is an important area theoretically 
as well as practically, requiring the cooperation of 
a number of disciplines represented in part in the 
Section on Aging. 

All the above areas have a common context — 
one which I emphasized in last year's annual re- 
port. I refer to the central concern with 
underlying capacity and potentiality of the or- 
ganism, whether it be the residual functions of the 
aged or the aberrations of the schizophrenic 
which throw light on the ordinarily unnoticed 
range of capacities in childhood and adulthood. 

Against this general background I shall first 
consider the work of the three sections in the 
Basic Research area: Aging, Animal Behavior, 
Learning and Perception, and then go on to con- 
sider the three in Clinical Investigations: Child 
Development, Personality, and Section of the 
Chief. 

SECTION ON AGING 

The general goal of the Section is to observe 
aging in both animals and humans with the aid of 
experimental methods in order to reveal and char- 
acterize mechanisms of biological and psychologi- 
cal interaction of general scientific importance. 
Through the research and theoretical efforts of 
staff members, the subject matter has undergone 
what in retrospect seems to have been a rapid tran- 
sition from a descriptive naturalistic phase into a 
more tightly organized state of knowledge. 
Knowledge and study of aging now seem to be 
established as complementary to early develop- 
ment as one of the fundamental scientific orienta- 
tions. 

The research program, while it has several 
facets, has two major foci. These are: (1) the 
gathering of systematic information about the be- 
havioral changes of aging, and (2) the psycho- 
biology of aging. Of continuing concern has been 
the analysis of age changes in learning and in 
speed of psychological processes, in which areas 
several advances were made. Cellular components 
and extracellular relations in the nervous system 
continued to be studied, with reason to hope for 
eventual success in identifying some of the bio- 
logical accompaniments of the behavioral changes 
of aging. 

During the past year the Section on Aging was 
engaged in a major activity in organizing and edit- 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



201 



ing the Handbook on Aging and the Individual: 
Psychological and Biological Aspects. Chapters 
for the Handbook were prepared by staff members 
on the subject matter of their research. The ad- 
vances in the state of our knowledge about aging 
has been made manifest by the completion of the 
Handbook. The organization of this material in 
many ways seemed a necessary early step in sys- 
tematic research effort. 

The data obtained in the extended study of 
healthy elderly men has been analyzed and draft 
manuscripts have been prepared reporting the re- 
sults. Data was gathered on 27 men over 65 years, 
all of whom were community residents and judged 
to be physiologically normal, "healthy." Also 
studied was a sample of 20 men who had asympto- 
matic or subclinical diseases (mostly vascular). 
Comparison of these two samples, and of these 
with data from provious studies of young men, 
gives considerable insight into what psychological 
changes may be regarded as the normal expect- 
ancy of advancing age and those changes fre- 
quently associated with disease states in older per- 
sons. It is expected that the manuscripts will be 
combined with those from investigators in other 
laboratories in the form of a monograph. The 
monograph will also report the intercorrelation of 
the experimental psychological, physiological, 
psychiatric and social psychological data. 

In general the results showed that the popula- 
tion studied compared more favorably with test 
results of young subjects than do the aged men 
of previous studies less well selected for health 
status. Two points may be made: (1) healthy 
men over 65 do better on psychological tests than 
men unselected for health and (2) age differences 
in patterns of abilities were found even in a popu- 
lation devoid of apparent disease. These findings 
are regarded as significant since they clearly point 
to the fact that age differences in psychological 
measurements may not be solely attributed to ill- 
nesses which frequently occur in aged individuals. 

Dr. Alfred D. Weiss reports the results of his 
measurements of hearing loss, click perception, 
diotic and dichotic digit span and response to de- 
layed speech feedback. The click discrimination 
measurements indicate that healthy older men do 
not differ from healthy young men in two click 
discrimination and that the less-healthy old tend 
to have larger discrimination times, i.e., a longer 



length of interval was required for discrimination 
between one or two clicks. While all auditory in- 
tensity measurements were correlated, none cor- 
related with the two-click discrimination thres- 
hold. Subjects were also presented with trains 
of clicks varying in number from one to ten at 
several rates of speed. The older subjects differed 
from young controls in their ability to enumerate 
the number of clicks. This measure of "percep- 
tion" did not correlate with intensity thresholds. 
In the digit-span measurements, distinct age de- 
clines were found. The older subjects were rela- 
tively poorer in the dichotic situation where they 
receive different digit series in the two ears. This 
suggests that the old may have more difficulty 
than the young in simultaneously monitoring two 
channels compared with monitoring a single 
channel. In the relayed speech feedback measure- 
ments very little difference was found between 
groups. 

Dr. James E. Birren, in collaboration with Drs. 
Botwinick and Weiss and Mr. Donald Morrison, 
analyzed the intercorrelations of the 23 cognitive 
and psychomotor variables and 9 audition vari- 
ables utilized in this study. The 32 variables were 
all intercorrelated and analyzed using Hotelling's 
Principal Component Method. About 58 percent 
of the common variance was accounted for on the 
basis of the first five components. Five independ- 
ent component scores were then derived for each 
subject. While all five scores showed mean differ- 
ences in favor of the "healthy" group, only com- 
ponent I was statistically significant. Component 
I was interpreted to be a measure of previously 
organized information. Interpretation of the 
finding was that late life illnesses result in a loss 
of stored information, primarily verbal; the 
healthy aged had higher mean information scores 
than would be expected in a young adult popula- 
tion. However, on perceptual and manipulative 
types of tasks, the older subjects were slower than 
the young. In the present study the health differ- 
ence was smaller than the age difference. This 
leads to an unanswered question of why "aging" is 
more important in speed of psychological func- 
tions and "health" more important in measures of 
stored verbal information. 

Dr. Ruth M. Riegel, Visiting Research Associ- 
ate of the Section, completed a comparison of 
factorial studies of the Wechsler-Bellevue, the 



202 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



Wechsler Adult Intelligence Scale of the German 
translation of the test HAWIE. Her results in- 
dicated that there seem to be no essential differ- 
ences in factorial structure of the different versions 
of the tests or between results obtained on German 
and American populations. 

Dr. Edward Jerome has been studying higher 
cognitive processes in aging subjects using the 
Logical Analysis Device of the Psychological Cor- 
poration and some additional classical problem- 
games. To date about 15 young and 10 elderly 
subjects have been studied in a sequence of in- 
dividual appointments which for each subject ex- 
tends over five or more days. The older subjects 
have expressed a preference for the automatically 
controlled device despite the fact that they experi- 
ence difficulty in its solution. The records are be- 
ing analyzed to identify the source of their diffi- 
culties in problem solution which in some cases 
reflects a failure to secure pertinent information 
to solve a problem or a disorderliness in the search 
for information. 

In related studies of learning and transfer, using 
the rat, a group of 40 additional animals were 
trained during the current year on two problems 
of different difficulties. The major findings may 
be summarized as follows: (1) senescent rats 
learned as quickly and efficiently as "mid-life" rats 
on a set of problems representing a wide range of 
difficulties, (2) senescent rats showed as much posi- 
tive transfer of training as did younger rats, and 
(3) no age differences were found in the degree of 
behavioral stereotypy, or rigidity of performance. 

Dr. Jack Botwinick found that when elderly 
and young adult subjects were compared with re- 
spect to performance on card sorting tasks (varied 
in: extent of perceptual matching or searching, 
number of stimulus aspects needed to be kept in 
mind and manipulated simultaneously, and re- 
learning requirements) it was found that the older 
subjects did relatively poorer with tasks that in- 
volved most mental manipulation and perceptual 
searching. Poor performance was also seen when 
age differences in motor or movement performance 
were examined. Relearning rate and practice 
effects were similar for the elderly and younger 
subjects. 

It was also found that elderly subjects, as com- 
pared with young, required more time to prepare 
or to organize for response. When time intervals 



were presented regularly but varied between 0.5 
and 4 seconds, a disproportionate slowing with age 
occurred with shortest interval. Much of this 
slowing was decreased with practice suggesting 
that at least part of the change with age is related 
to a difficulty in adjusting or learning to prepare 
for response during brief time intervals. Increas- 
ing the number of stimulus response alternatives 
had little, if any, differential age effect. 

Another study involved general level of activa- 
tion or reactivity in relation to age. There is 
reason to think that one aspect of general respon- 
siveness is measured by the GSR. Older adults 
were found to condition less readily and extin- 
guish more readily than younger adults. This 
lowered frequency of GSR output was taken as an 
index of reduced reactivity with age. 

Dr. Birren in collaboration with Dr. Klaus F. 
Riegel designed a study to examine the relations 
of psychomotor slowing and possible age changes 
in verbal association strengths and other language 
functions. In this study, subjects are presented 
with a variety of stimuli on a standard apparatus, 
the Psychomet, which permits measurements of 
individual response times while varying "key- 
light" relationships. Old and young subjects are 
being systematically observed in a series of tasks 
involving the measurement of simple movement 
times, simple and choice reaction time, and a 
variety of symbolic (numbers, letters, colors) and 
syllable and word associations. The general ob- 
jective of the study is to determine the extent to 
which certain of the age changes in behavior are 
the result of habit and overlearned language usage 
or in endogenous changes in the central nervous 
system which might be reflected in such aspects of 
performance as speed of response and perception, 
but which may have some more pervasive implica- 
tions for verbal reasoning. 

Dr. Eugene Streicher has made extracellular 
space determinations for brain. To obtain a meas- 
ure of the extracellular space of rat brain, the dis- 
tribution of thiocyanate between blood and brain 
was ascertained. (Thiocyanate is considered to 
be impermeable to most cells. ) The "thiocyanate 
space" was found to be a function of dosage, and 
extrapolation of the curve relating extracellular 
volume to plasma level suggests a space of approx- 
imately 5-10 percent. This figure is in general 
agreement with the estimates of electron micro- 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



203 



scopists, and indicates that, in contrast to liver or 
muscle, a large fraction of brain sodium and 
chloride is localized intracellularly. This conclu- 
sion was supported by measurements of brain 
extracellular space in rats in which cerebral edema 
had been experimentally induced. In this instance 
no change was observed in the distribution of thio- 
cyanate, although the sodium content of the brain 
was elevated 50 percent and that of the spinal cord 
was doubled. A few experiments were conducted 
with the congenitally jaundiced mutant strain of 
Wistar rat. In animals exhibiting neurological 
symptoms, such as difficulty in walking, the extra- 
cellular space of the spinal cord was decreased. 
The brain-thiocyanate space of 2y 2 -year-old rats 
was not significantly different from that of young 
animals indicating that possible brain changes in 
aging such as loss of cells, dehydration, gliosis, 
etc., if they do occur in rats, are not reflected in 
alterations of the extracellular space. However, in 
preliminary experiments, it was found that thio- 
cyanate was eliminated from the brains of older 
animals more slowly than from the brains of 
younger rats. 

Dr. William Bondareff completed his analyses 
of cytological preparations made the previous year 
from old and young, fatigued, and control animals. 
The experiment had been designed to investigate 
whether intracellular deposition of lipofuscin pig- 
ment could be initiated in animals by a course 
of acute muscular fatigue and whether the amount 
of intracelluar pigment normally present in the 
neurones of old animals could similarly be in- 
creased. In both cases it can be definitely stated 
that if such short-term muscular fatigue, as re- 
sults when animals are forced to swim until 
exhausted, does cause an increase in intracelluar 
pigment, the increase is not readily detected by 
cytological methods. It is possible also that pig- 
ment produced by this means was not chemically 
equivalent to that normally present and hence 
was not demonstrated by the cytological techni- 
que employed, but it seems justifiable, however, 
to conclude that there is no increase in intracellu- 
lar lipofuscin. 

Cytologic examination of the distribution of 
Nissl material does not indicate a great difference 
between the 3-month and 20-month animal. The 
Nissl pattern seen in 20-month animals is more 
closely related to that found in 3- than in 24- 
month-old animals. The cytological picture of 



20-month-old rats indicates that the metabolic 
processes of nucleic acid synthesis occur to a degree 
intermediate between that found in 3- and 24- 
month animals though more closely comparable 
to that of 3-month animals. 

SECTION ON ANIMAL BEHAVIOR 

Broadly speaking, research in the Section on 
Animal Behavior has been concerned with (a) 
defining the behavior served by association cor- 
tex in monkey, chimpanzee and man (b) deter- 
mining the neural mechanisms underlying this 
behavior and (c) specifying the neural mecha- 
nisms of attentive behavior in man. Accordingly 
studies have been designed to elucidate the inter- 
action within the association areas, and between 
these areas and the basal ganglia, hypothalamus, 
and brain stem. Participating in various aspects 
of these studies have been Dr. H. E. Rosvold, 
Chief of the Section, and Drs. M. Mishkin, A. F. 
Mirsky, M. K. Szwarcbart. Also participating 
have been Dr. Elinor Brush and Dr. Charles 
Butter, NIMH Post Doctoral Fellows; Dr. Karl 
Batting, a Visiting Scientist from Zurich; Dr. 
Stefan Brutkowski, a Visiting Scientist from War- 
saw; Dr. Bryan Robinson, a Research Associate; 
and Dr. H. Kuypers of University of Maryland, 
as a Consultant in Anatomy. 

Our earlier findings that, unlike monkeys, chim- 
panzees with damaged frontal lobes recover from 
an initial deficit on delayed-response problems 
has been confirmed in additional animals. Since, 
in a sense, the chimpanzee has a partical deficit 
in this behavior, he has been a particularly good 
subject in which to manipulate variables which 
may affect delayed-response behavior quantita- 
tively. Early results suggest, however, that it 
should be possible to specify which variables may 
be manipulated in the delayed-response test to in- 
crease its difficulty so that the chimpanzee's 
postoperative performance, like that of the 
monkey's, will be completely and permanently 
impaired. 

Behavioral studies with monkeys have also 
revealed something of the nature of the deficits 
following frontal-lobe damage. Findings last 
year suggested that monkeys with such damage 
are impaired in their ability to inhibit responses 
whether or not a delay is involved. More recent 
studies suggest that this impairment is quite gen- 



204 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



eral in the sense that frontal animals have difficulty 
in inhibiting any strong response tendency which 
develops as a result of training, preference, or 
novelty, and appears whether the response is de- 
pendent on visual, auditory, or tactual stimuli. 
An interpretation of these findings leads to the 
notion that in the frontal animal some on-going 
central process gets locked in and occludes others. 
Behavioral] y, the animal develops a set to respond 
and perseverates this set to the exclusion of others. 
Further research will attempt to set up critical 
tests of this notion. 

Earlier work based on limited information pro- 
vided by results on clelayed-alternation testing 
had suggested that the effects of frontal lobe 
lesions are similar to those of lesions in the head 
of the caudate nucleus. Research this year has 
extended the information to other types of delay 
problems, and to auditory, tactual, and visual 
learning tests. The results have been consistent 
in showing that the effects of caudate lesions, while 
quantitatively less severe, are qualitatively simi- 
lar to those of frontal lesions. Preliminary results 
of special histological studies using Nauta-Gygax 
silver staining procedures suggest that the behav- 
ioral similarities following damage to these two 
structures may be accounted for by the anatomical 
connections between them which have now been 
demonstrated. 

Last year we completed a study which appeared 
to implicate the splenium of the corpus callosum 
in delayed-alternation performance. More recent 
studies, however, indicate clearly that this is not 
so. The earlier effect appears to have resulted 
from damage either to the posterior columns of the 
fornix, the mid-line thalamic structures, or to 
some yet unsuspected structure. On-going re- 
search is attempting to determine which of these 
structures are in fact involved. 

Investigation of the role of the inferotemporal 
cortex in vision continues as a major effort of the 
section. A recent study in the literature questioned 
the specificity of the inferotemporal-visual rela- 
tionship, suggesting that not only inferotemporal 
but also other temporal neocortical areas serve 
visual functions, and that these areas serve not 
only visual but also olfactory functions. A special 
study undertaken to reinvestigate this problem 
provided no support for these suggestions, but 
confirmed instead the original thesis that the focal 



area for extrastriate visual processes is the infero- 
temporal area. 

It was hypothesized on the basis of earlier work 
that the inferotemporal area is connected with the 
primary visual area mainly, if not exclusively, via 
cortico- cortical connections. This conclusion de- 
rived from an experiment in which the cortico- 
cortical connections between an intact striate area 
in one hemisphere and an intact inferotemporal 
area in the opposite hemisphere were cut by the 
complete sectioning of the corpus callosum. This 
procedure produced an abrupt and severe decline 
in visual performance. An extension of this 
study, now in its early stages, demonstrates that 
the same impairment may be produced by section- 
ing the posterior third of the callosum only, 
whereas sectioning the anterior third is without 
effect on visual performance. On the basis of 
these results a detailed picture of a sensory-associ- 
ative visual system is beginning to emerge. Con- 
current anatomical studies, vising the Nauta tech- 
nique for staining degenerated fibers following 
selective cortical ablations, fully confirms this 
picture. 

The behavioral analysis of the visual impair- 
ment produced by inferotemporal lesions has de- 
pended until recently on work with monkeys 
trained by discrimination techniques. In an at- 
tempt to broaden the approach, new studies have 
now been initiated using generalization techniques 
and the studies designed to extend the analysis to 
chimpanzees and man are continuing. 

This year a new project has been developed to 
attack directly the problem of relating learning to 
brain function. The first step has been to establish 
base-line measures for continuous and periodic 
food-getting and water-getting responses in the 
monkey. The next step has been to compare these 
measures with those obtained by electrically 
stimulating through implanted electrodes the 
ventromedial and lateral hypothalamic areas 
(which are known to be reciprocally related in 
regulating eating and drinking) . The third step 
has been to study the effects on the electrical ac- 
tivity in these structures of food and water depri- 
vation and satiation. This phase of the study is 
in its very early stages, but preliminary results are 
encouraging. The next step, if the first two are 
successful, will be to study the changes in these 
measures as the learning of an alimentary condi- 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



205 



tioned response takes place in order to determine 
if a change in the activity of these brain centers 
occurs as the unconditioned and conditioned stim- 
uli become related in the process of learning. The 
long-range goal of this study is to gain an under- 
standing of the function of the hypothalamic 
hunger mechanisms in motivation and learning. 

The studies of attentive behavior in man during 
the past year have continued to involve detailed 
study of epileptic patients with presumed sub- 
cortical pathology and patients with electrodes im- 
planted in the temporal lobes. The abnormal elec- 
trical activity of the former group may interfere 
seriously with attentive behavior; that of the 
latter group does not. Approximately 15 patients 
with presumed subcortical pathology have been 
studied exhaustively from the standpoint of phasic 
interruptions in their ability to perform sustained 
vigilance tasks. Simultaneous with the behavior, 
the E.E.G. and seven different autonomic func- 
tions have been monitored. Present efforts are 
directed towards encoding the obtained informa- 
tion and analysis of the relationships among the 
several behavioral and physiological variables. 

Supplementing the study of neurological pa- 
tients has been the investigation of those condi- 
tions in normal individuals which produce 
impaired attentive behavior. Thus, behavioral, 
E.E.G. and autonomic variables have been studied 
in a group of ten normal controls under the influ- 
ence of prolonged sleep loss and of the drug 
chlorpromazine. 

The research conducted to date suggests certain 
broad similarities and striking differences among 
the effects of agents which impair attentive be- 
havior. Studies are currently being planned to 
test some of the anatomical-behavioral-autonomic 
relationships suggested by the information that 
has been obtained. 



SECTION ON PERCEPTION AND 
LEARNING 

A great deal of the data of the traditional psy- 
chophysical kind of experiment may reflect the 
operation of the observer's assumptions, attitudes, 
and language habits rather than elemental sensory 
processes from which perception is presumed to 
be elaborated. Instead, many perceptual phe- 
nomena which have in the past been interpreted 



as cognitive or judgmental may be more appropri- 
ately characterized as fundamental conditioning- 
adaptational processes not given directly in 
conscious experience. 

Dr. V. R. Carlson has utilized size constancy 
as a paradigm for exploring the usefulness of these 
considerations. This phenomenon refers to the 
empirical observation that perceived object size 
remains approximately invariant with variations 
in the distance of the object from the observer. 
It is a relationship which obtains under more or 
less natural environmental circumstances, but sys- 
tematic deviations occur when the relationship is 
studied experimentally. Two experiments in 
progress are concerned with determining whether 
these deviations have their explanation in terms of 
the attitudes and motivations which are inherent 
accompaniments of this kind of experimental 
situation. 

The first involves the effect of an instructional 
enhancement of the perspective attitude on size 
judgments. It appears clear that the degree of 
overconstancy which can be produced by attitudi- 
nal determinants is fully as great as the degree of 
underconstancy which can be produced by alterna- 
tive instructions. Underconstancy, however, has 
generally been interpreted in terms of underlying 
sensory mechanisms rather than as an attitudinal 
manifestation. 

The second size-constancy experiment investi- 
gates one aspect of the latter problem. LSD-25 
was used to produce a psychological state in which 
orientation of report toward subjective sensation 
is greatly enhanced. In this experiment, under- 
constancy has thus far occurred under circum- 
stances in which the effect cannot be interpreted 
as due to a change in basic sensory function. 

Two other perceptual studies are more directly 
concerned with the dependence of perceptual re- 
sponse on adaptation to various kinds of stimula- 
tion. Dr. Irvin Feinberg (Laboratory of Clinical 
Science) and Dr. Carlson have accomplished some 
initial work preliminary to determining relation- 
ships between kinesthetic and visual adaptation 
and to the measurement of adaptation to visual 
motion. 

The animal research program conducted at the 
Rockville Farm by Dr. John C. Calhoun and Dr. 
Barbehenn (postdoctoral fellow) has been directed 
chiefly this past year toward problems in which 



206 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



group size operates as a variable in the accommo- 
dation of the individual to a complex set of en- 
vironmental stimuli. 

Two long-term studies have been completed on 
the relationship of group size to learning in two 
kinds of social interaction problems. One of these 
situations required the proximity of two individ- 
uals in order for either to be rewarded ("coopera- 
tive" behavior) , while the other required that only 
one rat be present at the response situation for a 
reward to be received ("disoperative" behavior). 
The response was a lever press in order to obtain 
water. For cooperative behavior, smaller group 
size is associated with greater initial errors and 
a rapid rate of learning. Larger group size re- 
sults in fewer initial errors and a slower rate of 
learning. This trend arises from the fact that in 
the cooperative situation there is more opportunity 
for reward in a larger group purely on the basis 
of random activity. For disoperative behavior, 
the situation is different. The problem is learned 
but not rapidly enough to prevent gradual accrual 
of a deficit in water balance. A point is reached 
where the motivation to secure water or to respond 
to the lever-pressing situation cancels our prior 
learning and produces a situation in which in- 
dividuals stand side by side at the lever, even 
though this precludes the possibility of reward. 

It has been observed in several studies in which 
social groups of rats are maintained that wherever 
there is a high probability of two rats being in 
close proximity during occurrence of reward, the 
response situation develops a new definition which 
requires the presence of another individual. The 
other individual appears to serve as a secondary 
reinforcement, and few rats in the group will re- 
spond at those places or those times when other 
individuals are absent. Eventually such an altered 
pattern of response can lead to highly pathological 
social aggregations in the sense that the presence 
of other individuals in great numbers interferes 
with the execution of sequences of responses which 
form a behavioral unit. Where the behavior is 
instrumental in obtaining a physiologically neces- 
sary reward, this interference may lead to such 
an extreme deficit that most of the animals in the 
group die. 

Another observation is that there were clear 
alterations in the durations of behaviors and in 
the 24-hour cycles among individuals. This find- 



ing suggests a promising approach to further 
understanding of mechanisms of biological time. 
Dr. Barbehenn has studied the influence of litter 
size on later selection of places of habitation and 
places of visitation as these differentially offer 
opportunity for contact with other rats. Of 
several places to which rats had free access, those 
individuals who were members of small litters 
tended to seek out those situations in which contact 
with other individuals is minimal. Rats from 
litters of larger size tended to maximize opportu- 
nity for contact with other individuals. 

SECTION ON CHILD DEVELOPMENT 

The program of research in the Section on 
Child Development is concerned primarily with 
studying the characteristics of the infant under 
one year of age, and with those aspects of his 
environment that are likely to be significant in 
their effect on the infant's development and the 
formation of personality characteristics. 

Several current theories of personality develop- 
ment and of the etiology of mental pathologies, 
as well as of normal variations in personality and 
in other aspects of mental functioning, have em- 
phasized the importance of the infant's early ex- 
periences. We hear of the devastating effects of 
maternal deprivation and of environmental im- 
poverishment, and of such things as hostile rejec- 
tion and of overprotection of the child by his 
mother; we hear of the conditions which foster 
in the child feelings of security or insecurity, and 
of the effects of the child's early emotions and 
experiences on the course of his development. It 
becomes important, therefore, to explore in some 
detail just what is meant by these rather vague 
generalizations : Specifically, what behaviors and 
conditions does the infant in different settings 
experience, and just how does he react to them? 
How lasting are the effects of early experiences 
and how persistent are behaviors and response- 
tendencies that are learned (or at least manifested) 
in infancy? Can we determine which behaviors 
are species-specific, or within species genetically 
determined, and the differential effects of given 
experiences on children who are differently 
constituted ? 

Our studies take several forms, as they ap- 
proach a number of the different facets of these 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



207 



indicated conditions in the immature, developing 
organism as it interacts with different features 
in the environment. Within this framework, the 
nature of our investigations is determined to a 
considerable extent by the specific interests and 
preoccupations of the investigators involved. 

Dr. Harriet L. Rheingold has made one ap- 
proach by studying the comparative aspect of 
mother-infant interaction. She has so far made 
observations of maternal behavior and infant 
response in several mammalian species of widely 
differing degrees of complexity : the hamster, the 
dog, the monkey, and the human. In these com- 
parisons she will seek to differentiate aspects of 
maternal behavior specific to humans and those 
general across species. 

In the human infant, Dr. Rheingold has studied 
3-month-old infants in two widely different envi- 
ronments: first-born children in middle-class 
homes, and infants in an institution. She finds 
the same kinds of caretaking occurring in both 
environments, but great differences in amounts of 
these: the home baby has much more care, and 
care primarily by one person, whereas the insti- 
tution baby is left more to itself, but is cared for 
by many different people. At 3 months of age 
the behavior of the two groups of babies is about 
the same in many areas. The institution infants, 
however, proved to be more socially responsive 
to the examiner than the home infants. Sub- 
sequent research will attempt to uncover the 
causes of this difference. For example, were the 
institution infants more sociable because they 
were more deprived of stimulation, or because 
they did not discriminate the examiner as a 
strange person? 

Other studies of Dr. Bheingold's are relevant in 
working toward answers to these questions. These 
studies are in infant learning. She has, for ex- 
ample, shown that 3-month-old institution babies 
quickly learn to vocalize more frequently when 
their vocalizations are rewarded regularly by a 
smiling social response of an adult. As a result 
of these research efforts Dr. Rheingold has devel- 
oped some hypotheses about the genesis of social 
responsiveness and emotional attachment in the 
human infant. At a very early age there are al- 
ready developed in him both a responsiveness to 
stimuli in the environment and a searching of the 
environment for stimulation. The sight of some 



objects in his environment brings about smiles and 
vocalizations and other signs of delight. Of all 
the objects which arouse these responses the most 
potent appears to be the social object, that is, an- 
other human being. More than any other object 
the other human being brings to the child not only 
complex stimulation, but also stimulus change, 
and especially stimulation in response to the in- 
fant's own behavior. Because of the large role of 
vision in these interactions, Dr. Rheingold pro- 
poses the thesis that human sociability develops 
primarily from visual but also to some extent 
manipulatory exploratory behavior. 

Dr. Jacob L. Gewitz is also interested in the 
acquisition of social motives and attachments by 
the human infant. He believes that a large variety 
of stimuli can function as unconditioned postive 
reinf orcers of the infant's attachment behavior, in 
addition to those which meet the organic needs of 
the infant, and that reinforcing stimuli are likely 
to be provided also by nonhuman as well as human 
environmental changes. He plans to test these 
hypotheses by the technique of operant condition- 
ing, and has been developing an apparatus that is 
suitable for use with infants, to provide stimuli 
and record responses and thus measure the proc- 
esses of conditioning. He plans to continue these 
studies while on leave in Israel for an extended 
period. 

Another facet of the study of early personality 
development is that of variations in maternal be- 
havior. It is evident that normally a predomi- 
nant part of the infant's environment is furnished 
by the mother. Therefore, her predispositions, ex- 
pressed attitudes, and behavioral habits in regard 
to her infant may have a strong influence on the 
kinds of response habits the infant develops. Cer- 
tain types of maternal behavior have been given 
prominence in theories of the causes of, or at least 
strong causal components in, schizophrenia. Dr. 
Earl S. Schaefer has been working on a series of 
rating devices for use in classifying maternal at- 
titudes and behaviors. His current scales and 
hypotheses are outgrowths of the Parental At- 
titude Research Instrument and the Maternal Be- 
havior Research Instrument scales that he and Dr. 
Richard Q. Bell developed, and which have been 
described previously. In statistical analyses of 
the maternal behavior scales, Dr. Schaefer has 
f ound two factors and also a Guttman type of cir- 



208 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



cular order of neighboring that fit, not only the 
data from the Berkeley Growth Study, on which 
the scale was devised, but also a large number of 
other published data on maternal bahaviors. The 
two main orthogonal dimensions (i.e., factors) in 
this circumplex are autonomy-control and love- 
hostility. He has used this model, and the de- 
scriptions of maternal behaviors in the Berkeley 
study, as a basis for constructing a short-form 
Maternal Behavior Rating Scale and a Maternal 
Personality Rating Scale that can be used in 
evaluating the mother-child interactions as seen 
during a standard developmental testing situation. 
These scales are now being tried out, will be tested 
for reliability and validity, and revised on the 
basis of these preliminary trials. The relationship 
of these rated maternal variables will then be used 
to test out some hypotheses on mother-child re- 
lationships that Dr. Schaefer and Dr. Nancy Bay- 
ley have derived from the analyses of the Berkeley 
Growth Study data. For example, they found 
there some evidence that the lower-class mothers 
of boys were more punitive and authoritarian than 
the upper-class mothers, and that these relations 
in the mothers of girls were much less clear. 
There is also some evidence that the children of 
punitive, hostile mothers are more active and score 
higher on the developmental scales in the first 
year or two, but become less active and make 
poorer scores as they grow older. The happier, 
less excitable, inactive babies tend to be slow in 
their early development, and to have mothers who 
are more affectionate and generally accepting. 
There is also tentative evidence that the punitively 
controlling mothers have children who develop 
withdrawn, more maladaptive, kinds of social 
adjustment. 

Dr. Bell has conducted a series of researches 
based on evidence that the mother-child processes 
of interaction and the resultant maternal behavior 
and infant personality characteristics grow out of 
individual differences in the child as well as in the 
mother. He has observed and recorded the be- 
havior of 3-day-old infants, each for a 3-hour 
period with a standard set of stimuli. From a 
careful analysis of his data he has been able to 
derive five factors which differentiate newborn 
infants before they have had more than minimal 
experience with their mothers. These factors he 
has called : skeletal muscular strength, skin sensi- 



tivity (these two are negatively correlated with 
each other) , level of arousal, depth of sleep, and 
oral integration. In a further analysis of strength 
and skin sensitivity he finds both sex differences 
and within-sex variability in these factors. Males 
tend to be stronger, females more sensitive to skin 
stimulation. There appear, thus, to be genetic 
differences that are identifiable in the newborn 
infant. If these remain stable, they may well 
form a genetic basis for both individual and sex 
differences in personality variables. 

In following through with the research into 
other aspects of maternal characteristics as they 
affect the child, Dr. Schaefer is now testing out 
a scale of psychosomatic symptoms and psycho- 
logical reactions of women, before, during, and 
after pregnancy, and the relations of the latter 
two to difficulty of labor. In another approach to 
parent-child relationships he is testing out a scale 
for measuring children's perceptions of their par- 
ents' attitudes. Preliminary analysis show dif- 
ferences between normals, delinquents, and schizo- 
phrenics in their perceptions. 

The interaction of the infant with his environ- 
ment is determined to an important extent by the 
characteristics of the infant himself. Some of 
these have already been mentioned, such as indi- 
vidual differences in sensitivity to stimuli, and 
there are also evidences of differences in vulnera- 
bility to stress. Furthermore, the degree of de- 
velopment in an infant is a limiting factor both 
in his perceptions of his environment and in his 
ability to cope with the stimuli to which he re- 
sponds. It is therefore necessary to evaluate these 
factors in the studies of early personality and 
learning as they develop. With a view to improv- 
ing the tools for these evaluations, Dr. Bayley is 
revising and standardizing her mental and motor 
scales of infant development. These scales are 
designed to extend from 1 month through 30 
months of age. Another dimension which she is 
adding to the scales includes the appraisal of emo- 
tional and other reaction-tendencies that should 
prove useful as rough measures of the variables 
of sensitivity, vulnerability, and emotional tone, 
among others. At present, in cooperation with 
the NINDB collaborative projects, about half of 
the 1,500 standardization tests for the first 15 
months have been completed. With the use of 
such scales it will become possible to study learn- 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



209 



ing and evironmental adjustments in infants in 
relation to their developmental and "personality" 
scores. 

SECTION ON PERSONALITY 

The conduct and investigation of the process of 
psychotherapy continues to be one of the most 
fruitful sources of hypotheses for members of this 
section. This is illustrated in the work of Dr. 
Donald Boomer. He and Dr. Wells Goodrich have 
recently completed their replication of George 
Mahl's study of the relationship between instances 
of speech disturbances and global judgments on 
patients' anxiety level. It was found that (1) the 
appropriateness of the speech disturbance index 
as a general measure of anxiety for all subjects is 
doubtful ; (2) the therapist appears to be the best 
judge of anxiety in those patients who react to 
stress with some form of speech disruption; and 
(3) the reliability between judges concerning the 
identification of high and low anxiety phases is 
very poor. 

The significance of this research lies not only 
in the fact that the investigators have provided 
a more sophisticated analysis of the speech dis- 
turbance instrument, but that it has raised basic 
questions regarding the conceptualization of anxi- 
ety and its expression. Dr. Boomer now intends 
to test out some of his clinically derived hypoth- 
eses in the laboratory. He will attempt, under 
controlled conditions, to extend the investigation 
of tension indicators to include not only speech 
disturbances but other measures of autonomic 
lability. Issues of preferred modalities and the 
interrelationships among modalities under speci- 
fied conditions will be investigated. Further 
study of the phenomena of brief intercurrent 
speech pauses will be made to determine the part 
they play in what Mahl now defines as speech 
disturbances. It is to be noted that Dr. Goodrich's 
participation in this project has been reduced 
with his assumption of new administrative 
responsibilities. 

Drs. Joseph Handlon and Morris Parloff also 
have utilized their experience in group and indi- 
vidual therapy in their work concerning families 
of schizophrenics. This experience was relevant 
to the following activities : ( 1 ) The development 
of a test instrument devised to distinguish pat- 
terns of inter-family relationships which may 



correlate with mental health of the family mem- 
bers. (2) Dr. Handlon prepared a paper in which 
he derived and organized assumptions and hy- 
potheses implicit in existing theories of schizo- 
phrenia. This paper makes clear the implications 
for research strategy of the various concepts cur- 
rently in vogue regarding schizophrenia. (3) Dr. 
Parloff was coauthor of a paper read at the Ameri- 
can Psychiatric Association concerning recent 
experience with family group therapy. (4) Drs. 
Handlon and Parloff are currently preparing a 
paper to be delivered at the annual meeting of 
the American Group Psychotherapy Association 
contrasting group therapy and family group 
therapy. 

Dr. Handlon has taken an increasingly active 
role in a research program of the Section on Psy- 
chosomatic Medicine in the Adult Psychiatry 
Branch entitled, "Relation of Emotional Behavior 
and Adrenal Function." He has participated in 
the planning and execution of a study which in- 
volves observing changes in emotional state in re- 
sponse to three critical situations: (1) adaptation 
to a new environment (Clinical Center) ; (2) 
spontaneously occurring events of personal sig- 
nificance in the life of each subject; and (3) stand- 
ard commercial films that effectively present com- 
mon human problems. Two of the most signifi- 
cant findings to date related to the work of Dr. 
Handlon are the following: (1) the identification 
of two Rorschach patterns that correlate with two 
patterns of endocrine responses and (2) the identi- 
fication of individual affective response patterns to 
specified situational stresses. The technique of 
the self -rating questionnaire also assisted the in- 
vestigators in focusing their interviews and ward 
observations. 

The area of research in creativity is becoming a 
central concern of the Section. In general, the 
ultimate aim of the research will be to describe the 
conditions, internal and external, which are neces- 
sary and sufficient for productive thought. Our 
more immediate aim will be to learn about basic 
psychological processes involved in creative think- 
ing. One of the basic assumptions made in the 
work thus far is that the process is qualitatively 
the same at all levels of productive thinking and 
therefore can be studied not only in individuals 
designated as "creative" by their peers but also in 
generally talented subjects. It is the intention to 



210 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



attack the problem from two directions: (1) To 
undertake intensive clinical investigations with 
(a) scientists who have been designated as "crea- 
tive", and (b) individuals who have demonstrated 
talent but as yet have failed to be judged as crea- 
tive. Hopefully this will provide hypotheses for 
experimental study. (2) To select for intensive 
study basic psychological processes believed rele- 
vant to creative thinking. 

To date this section has undertaken four ap- 
proaches to the investigation of creativity. 

(1) The testing and interviewing of talented 
individuals. The sample has included Kesearch 
Associates selected for the NIH training program. 
Efforts have been made to integrate the current 
testing program with that of Dr. Morris Stein in 
the hope that this will facilitate comparison and 
interpretation of the findings. 

(2) The testing of the hypothesis that intuitive 
thinking is a significant element in creative 
thought. To this end, an instrument for measur- 
ing "intuitive problem solving" has been devised 
and is currently being tested and improved. 

(3) An investigation of the hypothesis that 
problem solving may be enhanced under condi- 
tions of reduced inhibition. This is being con- 
ducted by use of the Kleitman technique of moni- 
toring the dreams of individuals who have been 
given a problem-solving task which they failed to 
solve before retiring. 

(4) A pilot study has been conducted regarding 
the role of social-influence factors involved in col- 
laborative problem-solving. The variables in- 
cluded are (a) degrees of respect for the opinion 
of others and (b) the degree of approval received 
from such individuals in the course of collab- 
orative efforts to solve open-end problems. 
Although the analysis of the data is as yet incom- 
plete, there is evidence to suggest that (1) ap- 
proval is an important condition in stimulating an 
individual to produce original and plausible ideas 
and (2) the less accepting the individuals are of 
each other the greater the impact of receiving 
approval or criticism. 

It is hoped that it will soon be possible to under- 
take the intensive psychoanalytic investigation of 
a highly gifted but as yet "uncreative" individual 
to provide clinical data about inhibition and other 
hypotheses relevant to this project. 



SECTION OF THE CHIEF 

The major areas of research in this Section re- 
main schizophrenia and psychotherapy. Another 
involves the psychological aspects of physical ill- 
ness. In the area of psychotherapy there is some 
overlap with studies in the Section on Personality. 

In addition to Dr. David Shakow, the members 
of the Section concerned with the problem of 
schizophrenia are Drs. David Rosenthal and Theo- 
dore Zahn. Dr. William Lawlor of Fordham Uni- 
versity and Dr. Rue Cromwell of Peabody College 
participated in this program during the summer. 
One continuing study by Dr. Shakow involves the 
analysis of a large body of already existing ex- 
perimental data on the psychology of schizo- 
phrenia as a basis for development of theory in 
this field. During the year further progress was 
made on the analysis of this material. A paper on 
discrimination reaction time and a paper on the 
tautophone are among the many studies in an ad- 
vanced degree of analysis. It is hoped that eventu- 
ally this work will result in several monographs 
on the psychology of schizophrenia. 

Besides the above, studies in schizophrenia car- 
ried out during the year fell into three areas: 
heredity, psychophysiological responsivity, and 
simple adaptive behavior (reaction time). 

In the area of heredity, Dr. Rosenthal has con- 
tinued to examine the relative contributions of 
heredity and life experience factors to the severity 
and forms of expression of schizophrenia. To 
deal with this problem he has carefully analyzed 
the relevant literature in this area and has mar- 
shalled strong evidence for the following points : 
There is a group of schizophrenics in whom 
heredity factors probably play a strong part. In 
males, the form of the illness is mainly catatonic 
and the illness is severe. There is a group of 
schizophrenics in whom hereditary factors play a 
minimal part or none at all. In males, the form 
of the illness is mainly paranoid and the illness is 
relatively mild in that onset is late and outcome 
non-deteriorating. In the major twin studies of 
schizophrenia, chronic patients have constituted 
the bulk of the large samples, a factor which tends 
to yield overestimates of concordance rates. In 
the major twin studies, there are differences be- 
tween investigators with respect to sampling 
methods, determination of index cases, and diag- 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



211 



nostic criteria. These findings, plus the probabil- 
ity that too many twins are classified as dizygotic 
who are probably monozygotic, suggest that the 
present estimates of concordance rates are not 
reliable. Differences in concordance rates between 
female and male pairs, and between same sex 
versus opposite sex pairs occur with respect to 
schizophrenia and mental illness generally when 
the relationship is in the nuclear family, but not 
when the relationship is avuncular. This finding 
provides strong support for psychological influ- 
ences playing a significant role in the manifesta- 
tion of schizophrenia. 

Continuing experimentation went on in the area 
of psychophysiological responsivity as measured 
by GSR and heart-rate reactions to repetitive pres- 
entations of simple stimuli. The purpose of these 
studies is to investigate the autonomic reactions of 
schizophrenics under conditions which are mini- 
mal both in the quality of the stimuli and in the 
demands put on the subject. By intragroup com- 
parisons of these autonomic measures with meas- 
ures of overt behavior assessed by both ratings 
and more objective means such as experimental 
tasks, and by intergroup comparisons of schizo- 
phrenics with normals on these measures, the 
attempt is being made to evaluate the contribution 
of differences in autonomic functioning or arousal 
to deficits in the behavior of schizophrenic pa- 
tients. This minimal stimulus-minimal response 
situation is to serve as a "basal" condition for fur- 
ther studies in which the affective quality and/or 
demands on the subject will be increased. 

Preliminary findings on the GSR data indicate 
that, with respect to normals, schizophrenic pa- 
tients give about the same number of specific re- 
sponses to 40 repeated presentations of an auditory 
or visual stimulus, but the normals start at a 
higher level and habituate more rapidly. In addi- 
tion, the patients give more nonspecific responses 
relative to specific responses, have a lower baseline 
resistance and give responses with less amplitude 
than do normals. Data on a previous sample of 
patients indicated a significant correlation between 
response frequency and "mental health" or "ego 
intactness". This was not replicated on the latest 
sample of patients, but "mental health" correlated 
negatively with baseline resistance and response 
magnitude. The data from the two samples in- 
dictate, therefore, lowered autonomic responsivity 



to external stimuli by patients with poorer "mental 
health," but each sample shows this via a different 
measure. 

The studies of reaction time are being continued 
in a variety of settings and under various experi- 
mental conditions in order to answer some impor- 
tant theoretical questions about factors relating to 
this response. 

A number of findings have come out of our most 
recent studies in this area. These are: (1) 
Severity of schizophrenic disorganization is more 
reliably measured by any of three different meth- 
ods than are other traits and behaviors of such 
patients, and this measure correlates highly with 
various aspects of their performance on tests and 
experiments. (2) When preparatory intervals 
(Pi's) are administered in regular series, increas- 
ing in duration from 1 to 10 seconds, or decreasing 
from 10 to 1 second, normal S's do equally well 
under both conditions. Schizophrenic S's show 
poorer R T with longer Pi's in the ascending 
series, but do poorly on all Pi's in the descending 
series. (3) When Pi's are administered irregu- 
larly, schizophrenic S's are more influenced by the 
previous PI than are normal S's, who are also in- 
fluenced by it. (4) R T of schizophrenic S's is 
better when S is given a verbal "Ready" signal as 
compared to when Pi's are programed auto- 
matically. (5) If two relatively short Pi's are 
administered irregularly so that the probability 
with which each PI occurs is predetermined, then 
for normal S's, R T for the short PI increases as 
its probability of occurrence decreases. (6) In a 
R T situation where S is permitted to select his 
own Pi's and where he is not, normal S's prefer the 
"autonomous" condition where they select their 
own Pi's, whereas schizophrenic S's prefer the 
"controlled" condition where the Pi's are deter- 
mined for them. (7) Preliminary findings suggest 
that normal S's have better R T's under the "au- 
tonomy" condition, whereas the R T's of schizo- 
phrenic S's are better under the "controlled" 
condition. (8) Normal S's who perceived their 
mothers as more protective tended to see events as 
externally controlled, but schizophrenic S's did 
not. But schizophrenic S's tended to see their 
mothers as more hostile than did normal S's. 

In collaboration with the Clinical Science Lab- 
oratory the effects of various precursors of sero- 
tonin and of low dosages of Marsilid on autonomic 



556044—60- 



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212 



ANNUAL REVIEW OF INTRAMURAL RESEARCH 



responsivity, reaction time, and ability to main- 
tain a set are being studied in schizophrenic pa- 
tients. The data are still being analyzed, but it is 
planned to relate changes in these measures pro- 
duced by the drugs to changes in behavior as 
measured by ward ratings, interviews, and other 
experimental techniques as well as to biochemical 
changes. 

In an attempt to explore some of the conditions 
influencing reaction time and the maintenance of 
sets in normals, a reaction-time experiment was 
carried out on normals who had been deprived of 
sleep for 48-52 hours. The results showed that 
at the beginning of the half-hour session, if the 
preparatory interval was short and the subject 
could control the onset of the trial, there was no 
impairment. If any of these conditions were not 
met, sleep loss produced an impairment of reaction 
time. 

Dr. Rosenthal has continued to carry the ad- 
ministrative responsibility for the general aspects 
of the scientific investigation of a group of identi- 
cal quadruplet schizophrenic girls who have been 
under study by investigators from various NIMH 
laboratories for a period of several years. A large 
amount of data in different areas has been ac- 
cumulated and considerable progress was made 
during this year in its collation and analysis. 
The integration, evaluation, and write-up of this 
data have been begun by various investigators. 
The psychological material is in final stage of 
analysis by Drs. Rosenthal and Parloff . 

At the general theoretical level, Dr. Shakow has 
revised the paper presented at national and inter- 
national meetings : "The Recorded Psychoanalytic 
Interview as an Objective Approach to Research 
in Psychoanalysis." This is an attempt to provide 
the general rationale for the approach used in the 
major psychotherapy project. As an outgrowth 
of the research group's activity in the analysis of 
the first hour of the therapy, Dr. Shakow drew 
up a systematic account of modes of adaptation, 
particularly modes of defense, as they reveal them- 
selves in the psychotherapeutic process. 

Dr. Dittmann has continued his microanalytic 
studies of communication in the therapy process 
through the investigation of foot movements and 
speech disturbances. Two samples were collected 
and analyzed, the first finding association between 



foot movement and speech disturbance at the .01 
level, the second finding no association. A third 
sample has been collected, and is now being ana- 
lyzed for speech disturbance. The variability in 
movement from interview to interview is so great 
as to cast doubt on its usefulness beyond a charac- 
teristic of the interview as a whole : the spread is 
from 1 to 54 in 18 interviews so far studied. Thus, 
while in those interviews with many movements, 
the occurrence of a movement may mean some- 
thing about the events within the interview, inter- 
views with very few movements may be best 
characterized as lacking information, not lacking 
in the sort of disturbance we are interested in. It 
also is very clear that in later interviews there are 
more movements than in earlier ones, the first two 
samples having been selected within the first 40 
interviews, and the third from 50 to 100. Work 
now in progress is sampling much later interviews 
for simple count of foot movements. 

Dr. Dittmann together with an assistant has 
also been involved in the indexing of the inter- 
views. About a dozen interviews have now been 
checked by them. This work is chiefly to develop 
a standard level of abstraction, but has the side 
purpose, of course, of amassing a list of topics 
and referents which may be categorized. The 
progress is such that after one or two more inter- 
views the two will be able to index an interview 
independently and check agreement. 

Another important facet of the work on psycho- 
therapy is one with which Dr. Bergman is con- 
cerned. During recent years he has been gradu- 
ally developing "A General Theory of 
Psychotherapy." He presented a short formula- 
tion of this theory at the annual meeting of the 
American Psychological Association this year and 
is now setting up the project for the clinical testing 
of the evolving hypotheses. Since counter-condi- 
tioning is a fundamental part of his theory, Dr. 
Bergman is at the same time planning a series of 
experiments in which the attempt will be made 
experimentally to counter-condition so-called 
"anxiety" stimuli. 

Another area of research being carried on in the 
Section is Dr. Kendig's study of psychological 
factors related to physical disease in association 
with various of the other Institutes and with some 
outside agencies. Her major project is on the self- 
concept and body-image as related to disease 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



213 



susceptibility and organ choice. In this study she 
is exploring attitudinal factors insofar as they af- 
fect health and longevity. She is particularly in- 
terested in early childhood attitudes which may be 
instrumental in determining the nature of the 
self -concept and the body-image, especially in re- 
lation to susceptibility to illness, organ choice, 
course and outcome of disease. Dr. Kendig has 
developed an elaborate detailed questionnaire or 
interview schedule which is aimed at uncovering 
the attitudes towards the self and the body incul- 
cated in early childhood, explicitly by direct in- 
struction and implicitly by the emotional climate 
of the home and family reactions to illness. After 
pretesting on two patient groups and on one group 
of normal controls, the scales have been drawn up 
so the data can be coded and treated quantita- 
tively. The coding of the interview schedule 
which has already passed through a number of 
forms has been completed and a weighted scoring 
system devised. The two notable developments of 
the past year have been the extension of the proj- 
ect to include the study of a group of leukemia 
patients (NCI) and the selection and interviewing 
of a group of "normal" controls. With the ap- 
proval of the Medical Board, NIH personnel are 
drawn upon for the selection of the latter. The 
immediate goal is to complete the interviews. To 
achieve the desired n of 50 rheumatoid arthritics 
and 25 leukemia patients, 10 more subjects must 
be obtained in each group and patients continue to 
filter in slowly. About 50 more controls, carefully 
matched for age, race, sex, education, occupation, 
and religion must also be selected and interviewed. 
Meanwhile arrangements have been made for the 
blind scoring of the protocols. As soon as the first 
25 arthritics and their controls are scored, analysis 
of the data can begin, the second 25 arthritic cases 
then serving as a replication of the first. 

Dr. Daniel E. Berlyne, here as a Visiting Scien- 
tist for a year, is assigned to this Section but 
actually involved in projects in several sections. 
He has only recently come so that his projects are 
in the planning or about-to-be-embarked-upon 
stage. They comprise the following: (1) The 
psychophysiological effects of conflict. Experi- 
ments would deal with these questions : (a) the ef- 
fects of conflict on autonomic measures, (b) the 
effects of conflict on E.E.G. measures, (c) the ef- 
fect of number of competing response-tendencies 



on reaction-time, (d) the effects of complexity and 
incongruity of visual stimuli on orienting re- 
sponses and autonomic measures. (2) As part of 
the creativity project of the Section on Personal- 
ity, Dr. Berlyne plans to do some experiments on 
motivational factors underlying creative thinking, 
especially on the role of conflict and uncertainty in 
intellectual (epistemic) curiosity. This may lead 
to work on aesthetic behaviour. (3) In association 
with Dr. Rheingold, Dr. Berlyne plans to work on 
the determinants of perceptual curiosity in human 
infants, especially the role of novelty and com- 
plexity. 



Laboratory of Socio-environmental 
Studies * 

The development of the Laboratory's program 
in 1959 has perhaps been most impressive in the 
area of family studies, both in previously under- 
taken and in more recently initiated studies. 
These researches touch upon substantive problems 
such as (a) the structuring of interpersonal re- 
lationships in selected samplings of normal fami- 
lies, (b) the influence of maternal employment 
upon the mother's attitudes and her performance 
of the maternal role, and (c) husband- wife com- 
munication and interaction in the period ante- 
cedent to the hospitalization of either spouse for 
mental illness. They also touch upon methodo- 
logical problems such as (a) the validity of retro- 
spective data on early parent-child relationships 
and (b) the development of observational tech- 
niques to supplement and cross-validate interview 
techniques for the study of family relationships. 
This concentration upon the description and 
analysis of family patterns (though by no means 
to the exclusion of other areas of research) is in 
part a reflection of the strategic importance of 
the family as the group through which broader 
social and cultural influences upon personality 
development and behavior are mediated, and in 
part a matter of phasing of the long-range Labo- 
ratory program. 

The Laboratory's ultimate goal is to delineate 
and study the social norms and processes which 
(a) influence the development of personality and 
the distribution of mental health and illness, (b) 

•Prepared by John A. Clausen, Ph. D., Chief. 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



affect the individual's ability to carry out normal 
family, occupational, or community responsibili- 
ties and activities, and (c) govern the ways that 
disturbed or ill persons are perceived, defined, 
and dealt with at various stages of the life his- 
tory. Research has indicated a number of signif- 
icant correlations between grossly classified social- 
cultural phenomena and the phenomena of mental 
health and illness, but the specific linkages remain 
for the most part obscure. For example, rates 
of treated schizophrenia are inversely related to 
social status in the United States and Great 
Britain. Parent-child relationships exhibited by 
families which contain schizophrenic patients ap- 
pear to differ from those of families with normal 
children; and family patterns also appear to 
differ by social-status groups. But our knowl- 
edge of the distribution of such family patterns 
in the general population is meager, as is our 
knowledge of the specific effects upon personality 
development of various combinations of family 
patterns. More systematic study and more ade- 
quate specification of what goes on in the normal 
family is necessary, then, as a prelude to more 
sophisticated investigation of the epidemiology of 
mental illness. Hence, at the present stage of our 
knowledge, methodological studies and systematic 
analyses of the intercorrelated networks of varia- 
bles that constitute family patterns seem especially 
strategic approaches to the pursuit of our long- 
range goals. 

Hand in hand with the description and analysis 
of social structure and interpersonal networks 
must go the development of more adequate 
theories of personality development. While the 
social scientist looks to the psychologist and the 
psychiatrist for aid in conceptualizing personality 
organization and dynamics, he has a special re- 
sponsibility for analyzing and formulating the re- 
lationships between social structure and person- 
ality development. If certain combinations of re- 
lationships and experiences lead to distinct be- 
havioral responses (delinquency, drug addiction, 
aggressive striving, etc.), linkages will require 
conceptualization of both the social-experiential 
input and the behavioral output. 

Thus far the Laboratory's program has been 
focusing on the analysis of the "input" variables, 
and for some years our primary efforts are likely 



to continue here. It may be noted in the descrip- 
tions below, however, that the research of Camp- 
bell and Yarrow on children's peer-group relation- 
ships, the new study by Burton on processes of 
internalization of standards and values in chil- 
dren, and Rosenberg's research on self-images in 
adolescence deal to a considerable degree with 
social behaviors and psychological processes on 
the "output" side. 

Before turning to consideration of the specific 
projects under way in the several sections, it may 
be noted that during 1959 the Laboratory was for- 
tunate in recruiting four well qualified younger 
social scientists representing a broad range of 
skills in social psychology, sociology, and anthro- 
pology. Their research is still largely in process 
of formulation or early phases of data collection, 
but each man comes with broad research goals well 
envisoned. 

OFFICE OF THE CHIEF 

During 1959 the field work on the long-term 
study of the impact of mental illness on the family 
was substantially completed, as was the coding of 
data relating to family status and functioning, and 
to the spouse's perceptions of the patient's be- 
havior antecedent to hospitalization. An initial, 
analytical report on the marital relationship 
traced the sequence of behavioral changes and the 
efforts of the spouse to communicate with the pa- 
tient and cope with the family crisis. The sharp 
difference between confronting physical illness and 
confronting mental illness in a spouse is pointed 
up by the fact that, in many of the families 
studied, the patient's symptomatology entailed a 
direct assault upon the marital ties as such ; con- 
flict or progressive alienation characterized the 
marital relationship after the onset of mental ill- 
ness even where the marital tie had been close and 
happy. Subsequent analyses will attempt to assess 
the consequences, for the family unit and for the 
patient, of various modes of defining and coping 
with the illness. 

The Laboratory chief attended the Fourth 
World Congress of Sociology in Stresa, Italy, and 
subsequently visited a number of social psychiatry 
research centers in Scandinavia, France, and 
Great Britain. Out of this experience will come 
a general examination of the interrelationships 



MENTAL HEALTH — NEUROLOGICAL DISEASES AND BLINDNESS 



215 



between theoretical orientations of psychiatry and 
the organization of the psychiatric profession, on 
the one hand, and the development of community 
mental health services and of psychiatric research 
programs, on the other. 

Dr. Melvin Ember, who joined the Office of the 
Chief in September, will apply the research tech- 
niques and concepts of the anthropologist to the 
study of social influence in interpersonal behavior. 

COMMUNITY AND POPULATION STUDIES 

The long-range interests of this Section include 
the study of social organization; social differen- 
tiation and dominant cultural emphases (values, 
life styles, etc.) as these influence the develop- 
ment of personality; the distribution of various 
forms of deviant or problematic behavior ; and the 
development or utilization of various techniques 
for coping with problematic behavior. 

Drs. Kohn and Clausen, and Miss Eleanor E. 
Carroll, have for the past 4 years been studying 
the relationship of social class to child-rearing 
values and practices in 400 Washington families, 
each with a child aged 10-11 years. Following 
the previously reported analyses of social class 
differences in values parents hold for their chil- 
dren and in the ways in which they exercise their 
authority over the children, the past year's work 
has focused on the patterns of limit setting and 
emotional support vis-a-vis the 10-year-old child. 
Of considerable potential significance is the find- 
ing that while mother-child and father-daughter 
relationships do not markedly differ by social 
status, the father-son relationship seems more 
often close and emotionally supportive in the mid- 
dle class than in the working class. Whether one 
considers reports of relatively objective phenom- 
ena such as the amount of time spent by the father 
with his son or more subjective feelings on the 
part of both parents and sons of closeness and 
understanding, the working-class father seems to 
be less involved with his son — at least at age 10 
to 11 — than does the middle-class father. This 
finding runs counter to previously accepted state- 
ments suggesting that the son of the working-class 
father identifies or feels closer to him because he 
can more readily comprehend the father's occupa- 
tional activities and because the father is more 
often at home. Further analysis of these data will 



examine other characteristics which seem to in- 
fluence the perception of authority and affectional 
patterns; consensus among mother, father, and 
child with reference to such perceptions; and the 
probable implications of these patterns for per- 
sonality development. An effort will be made to 
secure some additional data by direct observation 
in selected households. 

A second study, dealing with adults rather than 
children and the occupational realm rather than 
the family, attempts to look at the relationship 
of social structure to personality from quite a 
different perspective. Dr. Stephen Boggs has de- 
veloped and pre-tested a questionnaire for use in 
a study of the ways in which the meaning of a 
man's job and work career is related to his emo- 
tional and physical health. He is now in process 
of administering this questionnaire to a sample of 
180-200 men in their middle working years. 

Dr. Erwin Linn continued analysis of data on 
patient characteristics, treatment, and duration 
of hospitalization among functional psychotics 
first admitted to Saint Elizabeths Hospital in the 
District of Columbia from 1953 through the first 
half of 1956. Dr. Linn this year concentrated his 
attention on the question of whether there have 
been changes, following the advent of drug ther- 
apy, in the relationship between patients' social 
characteristics and the probability of their release 
from the hospital within 1 year of admission. 
Various factors — such as marital status, race, sex, 
job stability, education, rural vs. urban back- 
ground, and social mobility — which had been 
highly associated with the probability of release 
in the period before the advent of drug therapy, 
were found to be of diminished significance in the 
later period. 

Dr. Gordon Allen, the geneticist attached to this 
Section, has continued the analysis of the data 
collected in his twin study of mental deficiency. 
He has also served as genetics consultant to other 
laboratories and in connection with such consulta- 
tion has developed quantitative methods for the 
analysis of zygosity. 

SOCIAL DEVELOPMENT AND FAMILY 
STUDIES 

The work of this Section has been largely di- 
rected to two major areas of research on the 



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ANNUAL REVIEW OF INTRAMURAL RESEARCH 



family — methodological problems of data collec- 
tion and problems of extending and refining vari- 
ables conceptualizing parent, child, and family as 
a group. A third area has been the study of the 
impact of immediate settings outside the family 
upon family members. 

The particular problems tackled in the Section's 
current family research stem from a number of 
deficiencies of methodology in this field: (1) Re- 
search on parent-child relations is rarely done in 
the natural setting of the home and rarely deals 
directly with intrafamilial behavior; it relies 
heavily upon interview and questionnaire proced- 
ures. (2) The data are generally the reports of 
a peculiarly involved reporter, the mother. (3) 
Research often requires of the mother remarkable 
synthesizing and interpretative powers in provid- 
ing the interviewer with the data he desires; she 
is asked to give her modal parental behaviors, and 
to summarize highly complex and variable inter- 
actions and feelings extending over long and in- 
definite time periods. (Is the home permissive, 
is she strict in the exercise of control over her 
child, etc.?) (4) Research on child-rearing has 
been channeled into an unvarying set of variables 
which tend to dominate research designs. 

In 1958 research was begun on observational 
techniques in the natural family setting. It has 
continued in the past year. Practical and techni- 
cal factors in the observer role are being examined 
(gaining access to the family, developing appro- 
priate observer-observed relations, developing 
specific techniques of observing). In a small 
group of families, data was gathered on areas of 
authority, discipline, and responsibilities, using 
participant observations, interviews, and question- 
naires. Preliminary work with the data attempts 
to combine observational and interview data in 
ways which will make it possible to analyze disci- 
plinary processes as to philosophy of parent, ac- 
tions of parents, and what it is in the situation 
which translates (or prevents the translation of) 
philosophy into behavior. Further, efforts are 
being made to include dimensions of the child's 
definition of the situation and his participation in 
the process — in evoking parental response and in 
responding to parental actions. 

Much of our work on observational methods to 
date has been preliminary to extensive data- 
gathering and definitive project design. This ex- 



ploration will continue in the next year with the 
expectation that a design will be developed, com- 
bining technical data-gathering problems and a 
substantive problem of child development. Mr. 
Thomas Gillette and Dr. Yarrow have been en- 
gaged in this work. Dr. Roger Burton, who 
joined the staff this fall from the Laboratory of 
Human Development, Harvard University, will 
also be working on problems in this area. 

A project concerned with problems inherent 
in data-gathering which relies on mother's retro- 
spective reporting on child and family relations 
has progressed during the past year under the di- 
rection of Dr. John Campbell and Dr. Yarrow. 
The research questions we a